Virgilia Macias

High-resolution Fourier-transform infrared chemical imaging with multiple synchrotron beams

Conventional Fourier-transform infrared (FTIR) microspectroscopic systems are limited by an inevitable trade-off between spatial resolution, acquisition time, signal-to-noise ratio (SNR) and sample coverage. We present an FTIR imaging approach that substantially extends current capabilities by combining multiple synchrotron beams with wide-field detection. This advance allows truly diffraction-limited high-resolution imaging over the entire mid-infrared spectrum with high chemical sensitivity and fast acquisition speed while maintaining high-quality SNR.

Resolution of clonal origins for endometriotic lesions using laser capture microdissection and the human androgen receptor (HUMARA) assay

OBJECTIVE To determine the clonal origins of endometriotic lesions using laser capture microdissection and PCR-based HUMARA assay. DESIGN Molecular genetic study of human tissue. SETTING Molecular genetics laboratory in an academic setting. PATIENT(S) Twenty patients with endometriosis. Forty specimens of endometriotic lesions from these patients and one specimen of normal endometrium were analyzed. INTERVENTION(S) Laser capture microdissection was used to harvest epithelial cells from single and multifocal endometrial lesions from paraffin-embedded and frozen tissues, and their clonality was determined with the HUMARA assay. MAIN OUTCOME MEASURE(S) Polymerase chain reaction-based HUMARA assay of clonality. RESULT(S) Thirty-eight specimens were polymorphic and thus informative. Most specimens were monoclonal, as determined by the HUMARA assay. In four specimens of multifocal lesions, polyclonality was detected, but upon more refined microdissections and further analyses, we found that each focus was monoclonal individually. CONCLUSION(S) Previously reported polyclonality is very likely to be attributed to the pooling of multifocal lesions or contamination of normal tissues. These results suggest that endometriotic lesions were monoclonal in origin, and in the case of multifocal lesions, each focus originates monoclonally; hence, different foci have independent origins. The monoclonality of endometriotic lesions suggests that they may carry neoplastic potentials, and the apparent independent origins of multifocal lesions suggest that reconstruction of individual lesion histories may help us to understand the initiation and progression of endometriosis.

The Cooperative Prostate Cancer Tissue Resource: A Specimen and Data Resource for Cancer Researchers

Purpose: The Cooperative Prostate Cancer Tissue Resource (CPCTR) is a National Cancer Institute-supported tissue bank that provides large numbers of clinically annotated prostate cancer specimens to investigators. This communication describes the CPCTR to investigators interested in obtaining prostate cancer tissue samples. Experimental Design: The CPCTR, through its four participating institutions, has collected specimens and clinical data for prostate cancer cases diagnosed from 1989 onward. These specimens include paraffin blocks and frozen tissue from radical prostatectomy specimens and paraffin blocks from prostate needle biopsies. Standardized histopathological characterization and clinical data extraction are performed for all cases. Information on histopathology, demography (including ethnicity), laboratory data (prostate-specific antigen values), and clinical outcome related to prostate cancer are entered into the CPCTR database for all cases. Materials in the CPCTR are available in multiple tissue formats, including tissue microarray sections, paraffin-embedded tissue sections, serum, and frozen tissue specimens. These are available for research purposes following an application process that is described on the CPCTR web site (www.prostatetissues.org). Results: The CPCTR currently (as of October 2003) contains 5135 prostate cancer cases including 4723 radical prostatectomy cases. Frozen tissues, in some instances including patient serum samples, are available for 1226 cases. Biochemical recurrence data allow identification of cases with residual disease, cases with recurrence, and recurrence-free cases. Conclusions: The CPCTR offers large numbers of highly characterized prostate cancer tissue specimens, including tissue microarrays, with associated clinical data for biomarker studies. Interested investigators are encouraged to apply for use of this material (www.prostatetissues.org).

Practical aspects of planning, building, and interpreting tissue microarrays: The Cooperative Prostate Cancer Tissue Resource experience

This is a review of several new approaches developed at or adopted by the Cooperative Prostate Cancer Tissue Resource (CPCTR) to resolve issues involved in tissue microarray (TMA) construction and use. CPCTR developed the first needle biopsy TMA, allowing researchers to obtain 200 or more consecutive cancer sections from a single biopsy core. Using radiographs of original paraffin blocks to measure tissue thickness we developed a method to produce TMAs with a larger number of usable sections. The modular approach to plan TMA construction is also a novel concept wherein TMAs of different types, such as tumor grade TMAs, metastasis TMA and hormone refractory tumors TMA can be combined to form an ensemble of TMAs with expanded research utility, such as support for tumor progression studies. We also implemented an open access TMA Data Exchange Specification that allows TMA data to be organized in a self-describing XML document annotated with well-defined common data elements. It ensures inter-laboratory reproducibility because it offers information describing the preparation of TMA blocks and slides. There are many important aspects that may be missed by both beginners and experienced investigators in areas of TMA experimental design, human subjects protection, population sample size, selection of tumor areas to sample, strategies for saving tissues, choice of antibodies for immunohistochemistry, and TMA data management.

The Progress on Genetic Analysis of Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma (NPC) is a rare malignancy in most parts of the world, but is one of the most common cancers in Southeast Asia. Both genetic and environmental factors contribute to the tumorigenesis of NPC, most notably the consumption of certain salted food items and Epstein-Barr virus infection. This review will focus on the current progress of the genetic analysis of NPC (genetic susceptibilities and somatic alterations). We will review the current advances in genomic technologies and their shaping of the future direction of NPC research.

Prediction of Prostate Cancer Recurrence Using Quantitative Phase Imaging

The risk of biochemical recurrence of prostate cancer among individuals who undergo radical prostatectomy for treatment is around 25%. Current clinical methods often fail at successfully predicting recurrence among patients at intermediate risk for recurrence. We used a label-free method, spatial light interference microscopy, to perform localized measurements of light scattering in prostatectomy tissue microarrays. We show, for the first time to our knowledge, that anisotropy of light scattering in the stroma immediately adjoining cancerous glands can be used to identify patients at higher risk for recurrence. The data show that lower value of anisotropy corresponds to a higher risk for recurrence, meaning that the stroma adjoining the glands of recurrent patients is more fractionated than in non-recurrent patients. Our method outperformed the widely accepted clinical tool CAPRA-S in the cases we interrogated irrespective of Gleason grade, prostate-specific antigen (PSA) levels and pathological tumor-node-metastasis (pTNM) stage. These results suggest that QPI shows promise in assisting pathologists to improve prediction of prostate cancer recurrence.

A simple inexpensive method for the production of tissue microarrays from needle biopsy specimens: examples with prostate cancer.

The use of tissue microarrays has become an efficient method for the high-throughput analysis of tissues with molecular markers, yet these studies have not been used to leverage the limited materials present in needle biopsies of human tissues. The use of these biopsy tissues is crucial to study diseases in patients who are treated by nonsurgical methods such as radiation, chemotherapy, or palliative care. The authors present a simple, inexpensive method for using needle biopsy specimens in tissue microarrays. Using this process with prostate cancer specimens, the authors demonstrate that over 150 slides can be produced from a single area of cancer in a needle biopsy and that the length of the core involved by cancer in the needle biopsy determines the number of available tissue microarray slides. The authors also note the optimal number of samples (three) needed from a single patient biopsy to guarantee sufficient material for analysis and perform an immunohistochemical correlation between needle biopsy and surgical resection tissue microarray samples for the quantitative marker Ki-67. This process can be extended to any type of needle biopsy specimen, increasing the number of studies and potential use of these tissues as a practical reality.

Exploring DNA methylation changes in promoter, intragenic, and intergenic regions as early and late events in breast cancer formation

BackgroundBreast cancer formation is associated with frequent changes in DNA methylation but the extent of very early alterations in DNA methylation and the biological significance of cancer-associated epigenetic changes need further elucidation.MethodsPyrosequencing was done on bisulfite-treated DNA from formalin-fixed, paraffin-embedded sections containing invasive tumor and paired samples of histologically normal tissue adjacent to the cancers as well as control reduction mammoplasty samples from unaffected women. The DNA regions studied were promoters (BRCA1, CD44, ESR1, GSTM2, GSTP1, MAGEA1, MSI1, NFE2L3, RASSF1A, RUNX3, SIX3 and TFF1), far-upstream regions (EN1, PAX3, PITX2, and SGK1), introns (APC, EGFR, LHX2, RFX1 and SOX9) and the LINE-1 and satellite 2 DNA repeats. These choices were based upon previous literature or publicly available DNA methylome profiles. The percent methylation was averaged across neighboring CpG sites.ResultsMost of the assayed gene regions displayed hypermethylation in cancer vs. adjacent tissue but the TFF1 and MAGEA1 regions were significantly hypomethylated (p ≤0.001). Importantly, six of the 16 regions examined in a large collection of patients (105 – 129) and in 15-18 reduction mammoplasty samples were already aberrantly methylated in adjacent, histologically normal tissue vs. non-cancerous mammoplasty samples (p ≤0.01). In addition, examination of transcriptome and DNA methylation databases indicated that methylation at three non-promoter regions (far-upstream EN1 and PITX2 and intronic LHX2) was associated with higher gene expression, unlike the inverse associations between cancer DNA hypermethylation and cancer-altered gene expression usually reported. These three non-promoter regions also exhibited normal tissue-specific hypermethylation positively associated with differentiation-related gene expression (in muscle progenitor cells vs. many other types of normal cells). The importance of considering the exact DNA region analyzed and the gene structure was further illustrated by bioinformatic analysis of an alternative promoter/intron gene region for APC.ConclusionsWe confirmed the frequent DNA methylation changes in invasive breast cancer at a variety of genome locations and found evidence for an extensive field effect in breast cancer. In addition, we illustrate the power of combining publicly available whole-genome databases with a candidate gene approach to study cancer epigenetics.

Effect of Soy Protein Isolate Supplementation on Biochemical Recurrence of Prostate Cancer After Radical Prostatectomy: A Randomized Trial

IMPORTANCE Soy consumption has been suggested to reduce risk or recurrence of prostate cancer, but this has not been tested in a randomized trial with prostate cancer as the end point. OBJECTIVE To determine whether daily consumption of a soy protein isolate supplement for 2 years reduces the rate of biochemical recurrence of prostate cancer after radical prostatectomy or delays such recurrence. DESIGN, SETTING, AND PARTICIPANTS Randomized, double-blind trial conducted from July 1997 to May 2010 at 7 US centers comparing daily consumption of a soy protein supplement vs placebo in 177 men at high risk of recurrence after radical prostatectomy for prostate cancer. Supplement intervention was started within 4 months after surgery and continued for up to 2 years, with prostate-specific antigen (PSA) measurements made at 2-month intervals in the first year and every 3 months thereafter. INTERVENTION Participants were randomized to receive a daily serving of a beverage powder containing 20 g of protein in the form of either soy protein isolate (n=87) or, as placebo, calcium caseinate (n=90). MAIN OUTCOMES AND MEASURES Biochemical recurrence rate of prostate cancer (defined as development of a PSA level of ≥0.07 ng/mL) over the first 2 years following randomization and time to recurrence. RESULTS The trial was stopped early for lack of treatment effects at a planned interim analysis with 81 evaluable participants in the intervention group and 78 in the placebo group. Overall, 28.3% of participants developed biochemical recurrence within 2 years of entering the trial (close to the a priori predicted recurrence rate of 30%). Among these, 22 (27.2%) occurred in the intervention group and 23 (29.5%) in the placebo group. The resulting hazard ratio for active treatment was 0.96 (95% CI, 0.53-1.72; log-rank P = .89). Adherence was greater than 90% and there were no apparent adverse events related to supplementation. CONCLUSION AND RELEVANCE Daily consumption of a beverage powder supplement containing soy protein isolate for 2 years following radical prostatectomy did not reduce biochemical recurrence of prostate cancer in men at high risk of PSA failure. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT00765479.

Analysis of iron, zinc, selenium and cadmium in paraffin-embedded prostate tissue specimens using inductively coupled plasma mass-spectrometry

Formalin-fixed paraffin-embedded (FFPE) tissue specimens represent a valuable and abundant resource of pathologic material for various biomedical studies. In the present study, we report the application of high-resolution inductively coupled mass-spectrometry (ICP-MS) for quantification of Fe, Zn, Se and Cd in FFPE prostate tissue. These elements have a possible role in the development of prostate diseases: while Zn and Se are needed for a healthy prostate, Cd shows multiple toxic and carcinogenic effects. Excessive accumulation of Fe induces the production of highly reactive hydroxyl radical species, which may play a role in cancer etiopathogenesis. To assess whether the levels of these metals in the FFPE prostate tissue represent their original content, we compared their levels with those in the fresh tissue (on dry weight basis) in samples obtained from 15 patients. We found that in FFPE tissue, the recoveries of Se, Fe, Cd and Zn were progressively decreased, 97+/-11% (r=0.88), 82+/-22% (r=0.86), 59+/-23% (r=0.69) and 24+/-11% (r=0.38), respectively. Thus, the use of correction factors, determined as k=0.16 for Se, k=0.20 for Fe, k=0.27 for Cd and k=0.67 for Zn, is required to estimate the retrospective levels of these elements in the parental non-processed fresh (wet) prostate tissue. The technique used in this study enables the analysis of archival FFPE prostate tissue for the concentrations of Fe, Zn, Se and Cd to study association between the levels of these metals and prostate disease.