Development of a novel noninvasive adhesive patch test for the evaluation of pigmented lesions of the skin.

BACKGROUND: The accurate clinical assessment of melanocytic neoplasms is a challenge for clinicians. Currently, obtaining a biopsy specimen and conducting a histologic examination is the standard of care. The incidence of melanoma in white populations is high, resulting in a large number of biopsy specimens. OBJECTIVE: The objective of this study is to develop a noninvasive genomic method using mRNA to classify pigmented skin lesions as either benign or malignant. METHODS: An adhesive patch method was used to obtain cells from the surface of melanocytic lesions. mRNA was extracted and a genomic signature was formulated in a training set of benign and malignant melanocytic neoplasms and subsequently tested in a validation set. RESULTS: A 2-gene signature assessing the expression levels of CMIP and LINC00518 was able to differentiate melanomas from nevi in an independent validation set of 42 melanomas and 22 nevi with a sensitivity of 97.6% and specificity of 72.7%. LIMITATIONS: Larger and more diverse sets of melanomas and nevi are needed for additional validation of the molecular expression profiling in various subsets of melanocytic neoplasms. CONCLUSION: Our data suggest that mRNA molecular signatures can serve as a highly useful noninvasive method of differentiating melanoma from nevi and decrease the number of unnecessary biopsies.

Development of a multiplexed urine assay for prostate cancer diagnosis.

BACKGROUND: Several studies have demonstrated the value of DNA methylation in urine-based assays for prostate cancer diagnosis. However, a multicenter validation with a clinical prototype has not been published. METHODS: We developed a multiplexed, quantitative methylation-specific polymerase chain reaction (MSP) assay consisting of 3 methylation markers, GSTP1, RARB, and APC, and an endogenous control, ACTB, in a closed-tube, homogeneous assay format. We tested this format with urine samples collected after digital rectal examination from 234 patients with prostate-specific antigen (PSA) concentrations > or =2.5 microg/L in 2 independent patient cohorts from 9 clinical sites. RESULTS: In the first cohort of 121 patients, we demonstrated 55% sensitivity and 80% specificity, with area under the curve (AUC) 0.69. In the second independent cohort of 113 patients, we found a comparable sensitivity of 53% and specificity of 76% (AUC 0.65). In the first cohort, as well as in a combined cohort, the MSP assay in conjunction with total PSA, digital rectal examination status, and age improved the AUC without MSP, although the difference was not statistically significant. Importantly, the GSTP1 cycle threshold value demonstrated a good correlation (R = 0.84) with the number of cores found to contain prostate cancer or premalignant lesions on biopsy. Moreover, samples that exhibited methylation for either GSTP1 or RARB typically contained higher tumor volumes at prostatectomy than those samples that did not exhibit methylation. CONCLUSIONS: These data confirm and extend previously reported studies and demonstrate the performance of a clinical prototype assay that should aid urologists in identifying men who should undergo biopsy.

Molecular classification of human cancers using a 92-gene real-time quantitative polymerase chain reaction assay.

CONTEXT: Correct diagnosis of the tissue origin of a metastatic cancer is the first step in disease management, but it is frequently difficult using standard pathologic methods. Microarray-based gene expression profiling has shown great promise as a new tool to address this challenge. OBJECTIVE: Adoption of microarray technologies in the clinic remains limited. We aimed to bridge this technological gap by developing a real-time quantitative polymerase chain reaction (RT-PCR) assay. DESIGN: We constructed a microarray database of 466 frozen and 112 formalin-fixed, paraffin-embedded (FFPE) samples of both primary and metastatic tumors, measuring expression of 22,000 genes. From the microarray database, we used a genetic algorithm to search for gene combinations optimal for multitumor classification. A 92-gene RT-PCR assay was then designed and used to generate a database for 481 frozen and 119 FFPE tumor samples. RESULTS: The microarray-based K-nearest neighbor classifier demonstrated 84% accuracy in classifying 39 tumor types via cross-validation and 82% accuracy in predicting 112 independent FFPE samples. We successfully translated the microarray database to the RT-PCR platform, which allowed an overall success rate of 87% in classifying 32 different tumor classes in the validation set of 119 FFPE tumor samples. CONCLUSIONS: The RT-PCR-based expression assay involving 92 genes represents a powerful tool for accurately and objectively identifying the site of origin for metastatic tumors, especially in the cases of cancer of unknown primary. The assay uses RT-PCR and routine FFPE samples, making it suitable for rapid clinical adoption.

Secondary amyloidosis and gastrointestinal stromal tumors. A case report and discussion of pathogenesis.

A 69-year-old man presented with a malignant gastrointestinal stromal tumor associated with secondary amyloidosis. The tumor had classic features of a malignant gastrointestinal stromal tumor with interlacing fascicles and whorls of spindled cells, numerous and conspicuous mitotic figures, and extensive coagulative necrosis. The cells stained diffusely for CD117 (c-Kit), confirming the diagnosis of gastrointestinal stromal tumor. The spleen, 1 adrenal gland, and part of the pancreas were removed en block with the stomach. By microscopy, the spleen and adrenal gland were partially replaced with amyloid deposits confirmed by Congo red staining, electron microscopy, and immunohistochemistry. In contrast, neither the tumor nor the surrounding vasculature showed amyloid deposition. To our knowledge, this represents only the second case of systemic amyloidosis associated with a gastrointestinal stromal tumor. This case is unique in that extensive, diffuse amyloid deposits were observed in the spleen, adrenal gland, and liver.

Interleukin 4 receptor on human lung cancer: a molecular target for cytotoxin therapy.

Previous studies have demonstrated that human lung tumor cell lines express interleukin 4 (IL-4) receptors, and IL-4 can mediate modest to moderate antiproliferative activity in vitro and in vivo in animal models of human lung tumors. On the basis of these studies, IL-4 was tested in clinical trials; however, it showed little antitumor activity in lung cancer patients. In the present study, we examined the expression of IL-4 receptors (IL-4Rs) in lung tumor samples and normal lung tissues and tested whether an IL-4R targeted agent will have better antitumor activity in vitro and in vivo compared with IL-4. IL-4R expression was tested by immunohistochemistry in 54 lung tumor samples and normal lung tissues in a tissue array, by reverse-transcription PCR and Northern blot analyses in lung tumor cell lines. Cytotoxic activity of IL-4 cytotoxin [IL-4(38-37)-PE38KDEL], composed of a circular permuted IL-4 and a mutated form of Pseudomonas exotoxin (PE38KDEL) was tested by protein synthesis inhibition and clonogenic assays in seven lung tumor cell lines. Antitumor activity of IL-4 cytotoxin was tested in vitro and in immunodeficient animal models of human lung tumors. We observed that IL-4Rs are expressed at higher levels in situ in lung tumor samples compared with normal lung tissues and IL-4 cytotoxin is highly and specifically cytotoxic to lung tumor cell lines in vitro. Intratumoral and i.p. administration of IL-4 cytotoxin to immunodeficient mice with s.c. established human lung H358 non-small cell lung cancer tumors mediated considerable antitumor activity in a dose-dependent manner with the higher dose producing durable complete responses. On the other hand, H460 non-small cell lung cancer tumors expressing low levels of IL-4R did not respond to IL-4 cytotoxin therapy. Because IL-4 cytotoxin mediates its antitumor activity through IL-4R, and a variety of lung tumors expressed high levels of IL-4R, we propose testing the safety of this agent in patients with lung cancer.