Atypical cellular blue nevi (cellular blue nevi with atypical features): lack of consensus for diagnosis and distinction from cellular blue nevi and malignant melanoma ("malignant blue nevus").

The distinction of cellular blue nevi (CBN) with atypical features ["atypical" CBN (ACBN)] from conventional CBN and malignant melanomas related to or derived from CBN remains a difficult problem. Here, we report on the diagnosis of various cellular blue melanocytic neoplasms by 14 dermatopathologists who routinely examine melanocytic lesions. Three parameters were assessed: (1) for between rater analyses, we calculated interobserver agreement by the kappa statistic (regardless of whether the diagnosis was correct). (2) For each individual lesion, we reported whether a majority agreement (>50%) was reached and, if so, whether the majority agreed with the gold standard diagnosis, derived from standardized histopathologic criteria for melanoma, definitive outcome such as metastatic event or death of disease, or disease-free follow-up for > or =4 years. (3) For the individual pathologists, we calculated sensitivity and specificity for each type of lesion. The study set included 26 melanocytic lesions: (1) 6 malignant melanomas developing in or with attributes of CBN; (2) 11 CBN with atypical features and indeterminate biologic potential (ACBN); (3) 8 conventional CBN; and (4) 1 common BN. The kappa values for interrater agreement varied from 0.52 (95% confidence interval 0.45, 0.58) for melanoma to 0.02 (0.05, 0.08) for ACBN and 0.20 (0.13, 0.28) for CBN. The kappa for all lesions was 0.25 (0.22, 0.28). The pathologists' sensitivities were 68.6% (61.0%, 76.1%) for melanoma, 33.1% (21.0%, 45.2%) for ACBN, and 44.6% (29.0%, 60.3%) for CBN. The specificities were 65.7% (55.8%, 75.6%) for melanoma, 84.7% (77.3%, 92.2%) for ACBN, and 89.9% (82.7%, 97.1%) for CBN. Overall, greater than 50% of the pathologists agreed and were correct in their diagnosis 38.5% (10 lesions) of the time. There was a majority agreement, but with an incorrect diagnosis, another 26.9% (7 lesions) of the time. Six of the 7 majority agreements with an incorrect diagnosis were for ACBN lesions. In summary, the results of our study indicate that there is substantial confusion and disagreement among experienced histopathologists about the definitions and biologic nature of cellular blue melanocytic neoplasms particularly those thought to have atypical features ("atypical" CBN).

Gene expression profiling of human sarcomas: insights into sarcoma biology.

Sarcomas are a biologically complex group of tumors of mesenchymal origin. By using gene expression microarray analysis, we aimed to find clues into the cellular differentiation and oncogenic pathways active in these tumors as well as potential biomarkers and therapeutic targets. We examined 181 tumors representing 16 classes of human bone and soft tissue sarcomas on a 12,601-feature cDNA microarray. Remarkably, 2,766 probes differentially expressed across this sample set clearly delineated the various tumor classes. Several genes of potential biological and therapeutic interest were associated with each sarcoma type, including specific tyrosine kinases, transcription factors, and homeobox genes. We also identified subgroups of tumors within the liposarcomas, leiomyosarcomas, and malignant fibrous histiocytomas. We found significant gene ontology correlates for each tumor group and identified similarity to normal tissues by Gene Set Enrichment Analysis. Mutation analysis done on 275 tumor samples revealed that the high expression of epidermal growth factor receptor (EGFR) in certain tumors was not associated with gene mutations. Finally, to further the investigation of human sarcoma biology, we have created an online, publicly available, searchable database housing the data from the gene expression profiles of these tumors (http://watson.nhgri.nih.gov/sarcoma), allowing the user to interactively explore this data set in depth.

Molecular alterations in primary prostate cancer after androgen ablation therapy.

PURPOSE: After an initial response to androgen ablation, most prostate tumors recur, ultimately progressing to highly aggressive androgen-independent cancer. The molecular mechanisms underlying progression are not well known in part due to the rarity of androgen-independent samples from primary and metastatic sites. EXPERIMENTAL DESIGN: We compared the gene expression profiles of 10 androgen-independent primary prostate tumor biopsies with 10 primary, untreated androgen-dependent tumors. Samples were laser capture microdissected, the RNA was amplified, and gene expression was assessed using Affymetrix Human Genome U133A GeneChip. Differential expression was examined with principal component analysis, hierarchical clustering, and Student's t testing. Analysis of gene ontology was done with Expression Analysis Systematic Explorer and gene expression data were integrated with genomic alterations with Differential Gene Locus Mapping. RESULTS: Unsupervised principal component analysis showed that the androgen-dependent and androgen-independent tumors segregated from one another. After filtering the data, 239 differentially expressed genes were identified. Two main gene ontologies were found discordant between androgen-independent and androgen-dependent tumors: macromolecule biosynthesis was down-regulated and cell adhesion was up-regulated in androgen-independent tumors. Other differentially expressed genes were related to interleukin-6 signaling as well as angiogenesis, cell adhesion, apoptosis, oxidative stress, and hormone response. The Differential Gene Locus Mapping analysis identified nine regions of potential chromosomal deletion in the androgen-independent tumors, including 1p36, 3p21, 6p21, 8p21, 11p15, 11q12, 12q23, 16q12, and 16q21. CONCLUSIONS: Taken together, these data identify several unique characteristics of androgen-independent prostate cancer that may hold potential for the development of targeted therapeutic intervention.

Morphological characterization of the breast in Proteus syndrome complicated by ductal carcinoma in situ.

Proteus syndrome (PS) is a severe, variable, and rare disorder with asymmetric and disproportionate overgrowth of body parts, cerebriform connective tissue nevi, epidermal nevi, dysregulated adipose tissue, and vascular malformations. It is associated with benign and occasionally malignant tumors. We report the first case of ductal carcinoma in situ (DCIS) in a 28-yr-old woman with PS who underwent a mastectomy for asymmetric overgrowth. The cut surface of the tissue showed a discrete, white, lobulated, solid mass with multiple cysts with occasional small polypoid nodules. Microscopically, the tissue was characterized by neoplastic and non-neoplastic changes. The former consisted of multiple intraductal papillomas and low-grade intraductal papillary, solid, and cribriform carcinoma. The non-neoplastic changes were characterized by cysts of various sizes, lined by cuboidal or apocrine cells, focally with epithelial papillary proliferation; the lumens contained eosinophilic, mucicarmine-positive, and PAS-positive material. Variable ductal proliferation and periductal, peri- and intra-lobular fibrosis with loose fibrous connective tissue was present. The carcinoma was positive for ER, PR, CK7, and MIB-1 (40%), and negative for p53 and CK20 staining. We conclude that DCIS may be one of the tumors associated with PS and that the proliferative phenotype serves as an initiator for carcinogenesis. This case highlights the difficulty of recognizing small foci of carcinoma in an asymmetrical overgrowth of the breast in a young woman with PS.

Prospective molecular profiling of melanoma metastases suggests classifiers of immune responsiveness.

We amplified RNAs from 63 fine needle aspiration (FNA) samples from 37 s.c. melanoma metastases from 25 patients undergoing immunotherapy for hybridization to a 6108-gene human cDNA chip. By prospectively following the history of the lesions, we could correlate transcript patterns with clinical outcome. Cluster analysis revealed a tight relationship among autologous synchronously sampled tumors compared with unrelated lesions (average Pearson's r = 0.83 and 0.7, respectively, P < 0.0003). As reported previously, two subgroups of metastatic melanoma lesions were identified that, however, had no predictive correlation with clinical outcome. Ranking of gene expression data from pretreatment samples identified approximately 30 genes predictive of clinical response (P < 0.001). Analysis of their annotations denoted that approximately half of them were related to T-cell regulation, suggesting that immune responsiveness might be predetermined by a tumor microenvironment conducive to immune recognition.

Generation and analysis of melanoma SAGE libraries: SAGE advice on the melanoma transcriptome.

In this study, we generated three SAGE libraries from melanoma tissues. Using bioinformatics tools usually applied to microarray data, we identified several genes, including novel transcripts, which are preferentially expressed in melanoma. SAGE results converged with previous microarray analysis on the importance of intracellular calcium and G-protein signaling, and the Wnt/Frizzled family. We also examined the expression of CD74, which was specifically, albeit not abundantly, expressed in the melanoma libraries using a melanoma progression tissue microarray, and demonstrate that this protein is expressed by melanoma cells but not by benign melanocytes. Many genes involved in intracellular calcium and G-protein signaling were highly expressed in melanoma, results we had observed earlier from microarray studies (Bittner et al., 2000). One of the genes most highly expressed in our melanoma SAGE libraries was a calcium-regulated gene, calpain 3 (p94). Immunohistochemical analysis demonstrated that calpain 3 moves from the nuclei of non-neoplastic cells to the cytoplasm of malignant cells, suggesting activation of this intracellular proteinase. Our SAGE results and the clinical validation data demonstrate how SAGE profiles can highlight specific links between signaling pathways as well as associations with tumor progression. This may provide insights into new genes that may be useful for the diagnosis and therapy of melanoma.

Pharmacokinetics of paclitaxel administered by hyperthermic retrograde isolated lung perfusion techniques.

OBJECTIVE: Although paclitaxel is widely used as a systemic agent for the treatment of solid tumors, limited information is available concerning administration of this taxane by regional techniques. The present study was undertaken to evaluate the pharmacokinetics and acute toxicity of paclitaxel administered by hyperthermic retrograde isolated lung perfusion techniques to ascertain its potential for the regional therapy of unresectable pulmonary neoplasms. METHODS: Adult sheep underwent 90 minutes of retrograde isolated lung perfusion with escalating doses of paclitaxel and moderate hyperthermia using a protein-free, oxygenated extracorporeal circuit and a steady perfusion pressure of 14 to 16 mm Hg. An additional animal received paclitaxel by means of 1-hour central venous infusion. Paclitaxel concentrations in lung tissues, perfusates, and systemic circulation were determined by high-performance liquid chromotography techniques. Cytotoxicity of paclitaxel in cancer cells and in normal human bronchial epithelial cells was evaluated in vitro using 4, 5-dimethylthiazo-2-yl-25-dipagnyl tetrazolium bromide assays. Lung tissues were examined by hematoxylin-and-eosin techniques. RESULTS: Paclitaxel concentrations (maximum concentration and area under the plasma concentration time curve) in perfused tissues increased with escalating perfusate doses. Uptake of drug into lung parenchyma appeared saturable at high paclitaxel exposure; a substantial pharmacokinetic advantage was observed. Paclitaxel concentrations in systemic circulation were undetectable or exceedingly low after perfusion. Histopathologic examination of lung tissues harvested 3 hours after completion of isolated lung perfusion revealed no immediate toxicity, even at a paclitaxel exposure 20-fold higher than that achievable after 1 hour of intravenous administration at the maximum tolerable dose in human subjects. Moderate hyperthermia enhanced paclitaxel-mediated cytotoxicity 5- to 100-fold in cultured cancer lines. No paclitaxel toxicity was observed in cultured normal human bronchial epithelial cells after exposure to paclitaxel under normothermic or hyperthermic conditions. CONCLUSIONS: These data support further evaluation of paclitaxel administered by hyperthermic retrograde isolated lung perfusion techniques for the treatment of unresectable malignant pulmonary tumors.

Are en face frozen sections accurate for diagnosing margin status in melanocytic lesions?

To assess the diagnostic accuracy of margin evaluation of melanocytic lesions using en face frozen sections compared with standard paraffin-embedded sections, we studied 2 sets of lesions in which en face frozen sections were used for analysis of surgical margins (13 from malignant melanomas [MMs] and 10 from nonmelanocytic lesions [NMLs]). Routine permanent sections were cut after routine processing. The slides were mixed and coded randomly. Fifteen dermatopathologists examined the cases separately. Margin status was categorized as positive, negative, or indeterminate. Kappa statistics were calculated per dermatopathologist and per case. One case from each group was excluded because epidermis was not available in the routine sections. Of 330 evaluations (22 cases, 15 dermatopathologists), there were 132 diagnostic discrepancies (40.0%): 66 each for MM and NML (mean per case for both diagnoses, 6). In 9 instances (6.8%), the change was from positive (frozen) to negative (permanent) and in 43 (32.6%), from negative (frozen) to positive (permanent). There was poor agreement between frozen and permanent sections (kappa range per dermatopathologist, -0.1282 to 0.6615). If permanent histology is considered the "gold standard" for histologic evaluation, en face frozen sections are not suitable for accurate surgical margin assessment of melanocytic lesions.

Molecular differentiation of high- and moderate-grade human prostate cancer by cDNA microarray analysis.

The prognosis of men with moderate-grade prostate cancer is uncertain. At present, there are few if any reliable molecular markers that can distinguish moderate-grade tumors from those that behave more aggressively. To better understand the molecular basis of human prostate cancer and potentially provide information toward more accurate prognosis, we measured and analyzed gene expression profiles of 13 high- and moderate-grade human prostate tumors using cDNA microarrays. The expression of 136 genes was observed to differ significantly (P < 0.001) between normal prostate and tumors using one-sample t testing and Wilcoxon ranking. Hierarchical clustering of genes demonstrated a relatively similar pattern of differential expression across the tumors. However, importantly, permutation t tests (two-tailed P < 0.001) revealed 21 genes whose expression profiles segregated moderate- and high-grade tumors from each other, which was significantly (P < 0.03) greater than what was expected by chance. These results were compared in silico with prostate cancer profiling efforts performed by other groups, including a meta-analysis of four data sets, which validated many of the dysregulated genes. We suggest that these data provide insight into the molecular nature of clinically aggressive prostate cancer.

Parathyroid hormone-related protein and hypercalcemia in patients with metastatic melanoma: case report and review.

Hypercalcemia associated with malignancy has been attributed to osteolytic processes secondary to bony metastases and to humoral factors causing increased bone resorption and decreased renal excretion of calcium. Parathyroid hormone-related protein (PTH-rP) is a humoral factor that has been associated with hypercalcemia in renal cell carcinoma, squamous cell carcinoma, and bladder carcinoma. Hypercalcemia does occur in patients with melanoma; however, few studies have reported on hypercalcemia in these patients, and even fewer have described a direct connection to PTH-rP. We here report a patient with stage IV malignant melanoma presenting with severe hypercalcemia associated with elevated PTH-rP levels. Immunohistochemistry showed strong expression of PTH-rP in biopsy of the patient's subcutaneous masses. In addition, we found a 4.9% incidence of hypercalcemia in 1,146 consecutive patients treated for metastatic melanoma at the Surgery Branch of the National Cancer Institute between January 1, 1988 and March 31, 2000. Thus, PTH-rP may play a significant role in severe hypercalcemia in patients with metastatic melanoma. The discovery of PTH-rP and relevant literature will also be reviewed.

The effects of gp100 and tyrosinase peptide vaccinations on nevi in melanoma patients.

BACKGROUND: A new approach to prevent disease recurrence in high-risk melanoma patients involves immunization with gp100 and tyrosinase peptides. This is the first study to examine the effects of such treatments on nevi. DESIGN: We studied biopsies of 'clinically atypical' nevi from 10 patients before and after peptide vaccination. All had a cutaneous melanoma measuring at least 1.5 mm in depth, satellite metastases, or at least one positive lymph node. We performed immunohistochemical stains for CD3, CD4, CD8, MHC-I, MHC-II, CD1a, HMB-45, MART-1, tyrosinase, bcl-2, p53, and Ki-67 (mib-1). RESULTS: Immunohistochemistry showed no differences in staining due to vaccination in either the immunologic or melanocytic markers. However, there was a significant increase in both p53 and bcl-2 staining, and a trend toward decreased Ki-67 staining, in the nevi post-treatment. DISCUSSION: The primary goal of peptide vaccinations with gp100 and tyrosinase is to activate melanoma-specific T cells in order to prevent melanoma recurrence. Nevi were studied in order to assess the effects on benign melanocytes. No significant changes in lymphocytes, langerhans cells, expression of MHC antigens, or melanocytic markers were found. The increase in p53 and bcl-2 raises the possibility that vaccination with melanocytic antigens stimulates a response in benign melanocytes.

Invasion of human tissue ex vivo by Borrelia burgdorferi.

Borrelia burgdorferi sensu stricto is an etiological agent of Lyme disease. The lack of an adequate ex vivo system for human tissue infection is an obstacle to fully understanding the molecular mechanisms of invasion of tissue by B. burgdorferi and its adaptation within the human host. Here, we report on the development of such a system. We inoculated blocks of human tonsillar tissue with B. burgdorferi spirochetes, cultured them in a low-shear rotating wall vessel (RWV) bioreactor, and analyzed them using light and electron microscopy, nested polymerase chain reaction (PCR), and quantitative real-time PCR. Also, we evaluated the expression of the outer surface proteins (Osps) OspA and OspC by use of quantitative Western blotting. Light and electron microscopic analysis revealed multiple spirochetes localized extracellularly within the tissue, and their identity was confirmed by PCR. Quantification of spirochetes inside the RWV-cultured tonsillar tissue demonstrated that the number of B. burgdorferi exceeded the initial inoculum by an order of magnitude, indicating that spirochetes replicated in the tissue. Electron microscopic analysis showed that some spirochetes were arranged in cystic structures and that invading spirochetes differentially expressed surface proteins; both of these features have been described for infected tissues in vivo. The system we have developed can be used to study B. burgdorferi pathogenesis under controlled conditions ex vivo, in particular to explore the gene activation responsible for the adaptation of B. burgdorferi to human tissue that leads to Lyme disease.

Lyme-associated parkinsonism: a neuropathologic case study and review of the literature.

Neurological complications of Lyme disease include meningitis, encephalitis, dementia, and, rarely, parkinsonism. We present a case of striatonigral degeneration, a form of multiple system atrophy, in Lyme-associated parkinsonism. A 63-year-old man presented with erythema migrans rash, joint pains, and tremors. Serum and cerebrospinal fluid antibodies and polymerase chain reaction for Borrelia burgdorferi were positive. Clinical parkinsonism was diagnosed by several neurologists. Despite treatment, the patient continued to decline, with progressive disability, cognitive dysfunction, rigidity, and pulmonary failure. At autopsy, the brain showed mild basal ganglia atrophy and substantia nigra depigmentation, with extensive striatal and substantia nigral neuronal loss and astrogliosis. No Lewy bodies were identified; however, ubiquitin-positive glial cytoplasmic inclusions were identified in striatal and nigral oligodendroglia. There were no perivascular or meningeal infiltrates, the classic findings of neuroborreliosis. To our knowledge, this is the first report of striatonigral degeneration in a patient with B burgdorferi infection of the central nervous system and clinical Lyme-associated parkinsonism.

Metastatic nevoid melanoma in a 4 1/2-year-old child.

BACKGROUND: Childhood melanoma is a rare and controversial diagnosis. METHODS: We present the case of a 4 1/2-year-old child found to have an expanding, elevated pigmented lesion on her back. RESULTS: The biopsy showed a symmetrical, well circumscribed lesion. However, higher magnification revealed sheets of nevoid cells infiltrating deep into the dermis, lacking maturation, and exhibiting a high mitotic rate (average 5/10 high-power field) with deep mitoses. The possibility of nevoid melanoma was raised, and re-excision and sentinel lymph node (SLN) biopsy were recommended. Two SLNs were positive for melanoma, verified by immunohistochemical staining. In order to further characterize this melanoma, we performed immunohistochemistry for the tumor-suppressor p53, proliferation marker MIB-1, and oncogenes Bcl-2, cyclin D-1, and MDM-2. Staining for p53 was diffusely positive in the primary and the metastasis; MIB-1 showed moderate proliferative rate in the primary (approximately 10%); Bcl-2 was weakly positive in the primary and showed focal staining in the metastasis; cyclin D-1 was strongly positive in the primary and metastasis; and MDM-2 staining showed scattered positive cells in both lesions. CONCLUSIONS: These findings are consistent with a metastatic nevoid melanoma arising in the absence of predisposing disease in a young child, a distinctly unusual occurrence.

Determination of a minimal deletion interval on chromosome band 8p21 in sporadic prostate cancer.

Loss of the short arm of chromosome 8 is a common event in prostatic neoplasms. Previous studies indicate that there may be up to three separate tumor suppressor genes on chromosome arm 8p, based on patterns of allelic loss. The responsible gene or genes have yet to be identified. In the present study, we used laser-capture microdissection of primary human prostate tumors and 17 microsatellite markers across chromosome band 8p21 to determine a minimal deletion interval. From an initial set of 120 cases, three tumors contained overlapping interstitial deletions on chromosome band 8p21. The three cases define an internally consistent minimal candidate tumor suppressor gene interval of approximately two megabases.

Evaluation of ethanol-fixed, paraffin-embedded tissues for proteomic applications.

We previously reported that ethanol fixation and paraffin embedding of tissues produce excellent histomorphology and good preservation of macromolecules. Here, we present a detailed evaluation of ethanol-fixed tissues for proteomic initiatives. When proteins were extracted from ethanol-fixed, paraffin-embedded prostate tissue, resolved by two-dimensional gel electrophoresis (2-DE), and stained by standard methods, several hundred protein molecules could be detected and successfully analyzed by mass spectrometry. Protein profiles obtained from ethanol-fixed tissues were highly similar to those observed from frozen tissues, in contrast to the poor protein recovery from formalin-fixed material. The protein content of specific cells that were microdissected from ethanol-fixed tissue sections using laser capture microdissection could also be successfully analyzed by 2-DE. We observed that eosin staining of tissue sections had a detrimental effect on protein separation, whereas hematoxylin staining had minimal consequence. In order to illustrate the applicability of ethanol-fixed tissues for proteomic discovery studies, we compared the protein profiles of patient-matched, normal prostatic epithelial cells and invasive adenocarcinoma cells obtained from ethanol-fixed, paraffin-embedded tissues. A number of differentially expressed proteins was discovered and identified by mass spectrometry. Immunohistochemical analyses performed on ethanol-fixed tissue sections were in agreement with the proteomic discovery findings. In light of these results, we conclude that ethanol-fixed tissues can be successfully utilized for proteomic analyses.

Proteomic analysis of human prostate cancer.

Proteomics is a promising approach in the identification of proteins and biochemical pathways involved in tumorigenesis. In an effort to discover such proteins and pathways that are deregulated in prostate tumorigenesis, cellular proteomes of matched normal prostate epithelial cells and high-grade prostate cancer cells were analyzed by tissue microdissection, two-dimensional electrophoresis, and mass spectrometry. Forty protein alterations were detected in the tumors; however, the majority of these changes were not shared among the 12 neoplasms. In contrast, parallel cDNA microarray analysis identified a number of common gene expression changes. The marked heterogeneity of the observed protein alterations may have significance with regard to tumor biology and research strategies for molecular profiling analyses of human prostate cancer.

Cancer regression and autoimmunity induced by cytotoxic T lymphocyte-associated antigen 4 blockade in patients with metastatic melanoma.

Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is a critical immunoregulatory molecule (expressed on activated T cells and a subset of regulatory T cells) capable of down-regulating T cell activation. Blockade of CTLA-4 has been shown in animal models to improve the effectiveness of cancer immunotherapy. We thus treated 14 patients with metastatic melanoma by using serial i.v. administration of a fully human anti-CTLA-4 antibody (MDX-010) in conjunction with s.c. vaccination with two modified HLA-A*0201-restricted peptides from the gp100 melanoma-associated antigen, gp100:209-217(210M) and gp100:280-288(288V). This blockade of CTLA-4 induced grade III/IV autoimmune manifestations in six patients (43%), including dermatitis, enterocolitis, hepatitis, and hypophysitis, and mediated objective cancer regression in three patients (21%; two complete and one partial responses). This study establishes CTLA-4 as an important molecule regulating tolerance to "self" antigens in humans and suggests a role for CTLA-4 blockade in breaking tolerance to human cancer antigens for cancer immunotherapy.

Evaluation of non-formalin tissue fixation for molecular profiling studies.

Using a general strategy for evaluating clinical tissue specimens, we found that 70% ethanol fixation and paraffin embedding is a useful method for molecular profiling studies. Human prostate and kidney were used as test tissues. The protein content of the samples was analyzed by one-dimensional gel electrophoresis, immunoblot, two-dimensional gel electrophoresis, and layered expression scanning. In each case, the fixed and embedded tissues produced results similar to that obtained from snap-frozen specimens, although the protein quantity was somewhat decreased. Recovery of mRNA was reduced in both quantity and quality in the ethanol-fixed samples, but was superior to that obtained from formalin-fixed samples and sufficient to perform reverse transcription polymerase chain reactions. Recovery of DNA from ethanol-fixed specimens was superior to formalin-fixed samples as determined by one-dimensional gel electrophoresis and polymerase chain reaction. In conclusion, specimens fixed in 70% ethanol and embedded in paraffin produce good histology and permit recovery of DNA, mRNA, and proteins sufficient for several downstream molecular analyses. Complete protocols and additional discussion of relevant issues are available on an accompanying website (http://cgap-mf.nih.gov/).