Wilfrido D. Mojica

Elastofibroma dorsi : Elaboration of cytologic features and review of its pathogenesis

Elastofibroma is a slow‐growing soft tissue lesion characteristically found between the inferior scapula and chest wall. Because it behaves clinically in a benign manner, fine‐needle aspiration (FNA) represents the simplest and quickest method of obtaining a definitive diagnosis, thus obviating more invasive means of obtaining a tissue diagnosis. However, due to the nature of this lesion a correct diagnosis can inadvertently be missed. Herein we describe the findings of a recent FNA that obtained abundant diagnostic material and elaborate upon the spectrum of cytologic features of the elastic fibers that can be identified. These features should be recognized, since aspiration biopsy in elastofibromas can lead to hypocellular smears. In addition, we discuss recent developments in the pathophysiology of elastic fibers and their application toward understanding the generation of an elastofibroma. Diagn. Cytopathol. 2000;23:393–396.

Evidence for field effect cancerization in colorectal cancer

We compared transcript expression, and chromosomal changes on a series of tumors and surrounding tissues to determine if there is evidence of field cancerization in colorectal cancer. Epithelial cells were isolated from tumors and areas adjacent to the tumors ranging from 1 to 10cm. Tumor abnormalities mirrored those previously reported for colon cancer and while the number and size of the chromosomal abnormalities were greatly reduced in cells from surrounding regions, many chromosome abnormalities were discernable. Interestingly, these abnormalities were not consistent across the field in the same patient samples suggesting a field of chromosomal instability surrounding the tumor. A mutator phenotype has been proposed to account for this instability which states that the genotypes of cells within a tumor would not be identical, but would share at least a single mutation in any number of genes, or a selection of genes affecting a specific pathway which provide a proliferative advantage.

Normal colon epithelium: a dataset for the analysis of gene expression and alternative splicing events in colon disease

BackgroundStudies using microarray analysis of colorectal cancer have been generally beleaguered by the lack of a normal cell population of the same lineage as the tumor cell. One of the main objectives of this study was to generate a reference gene expression data set for normal colonic epithelium which can be used in comparisons with diseased tissues, as well as to provide a dataset that could be used as a baseline for studies in alternative splicing.ResultsWe present a dependable expression reference data set for non-neoplastic colonic epithelial cells. An enriched population of fresh colon epithelial cells were obtained from non-neoplastic, colectomy specimens and analyzed using Affymetrix GeneChip EXON 1.0 ST arrays. For demonstration purposes, we have compared the data derived from these cells to a publically available set of tumor and matched normal colon data. This analysis allowed an assessment of global gene expression alterations and demonstrated that adjacent normal tissues, with a high degree of cellular heterogeneity, are not always representative of normal cells for comparison to tumors which arise from the colon epithelium. We also examined alternative splicing events in tumors compared to normal colon epithelial cells.ConclusionsThe findings from this study represent the first comprehensive expression profile for non-neoplastic colonic epithelial cells reported. Our analysis of splice variants illustrate that this is a very labor intensive procedure, requiring vigilant examination of the data. It is projected that the contribution of this set of data derived from pure colonic epithelial cells will enhance studies in colon-related disease and offer a vital baseline for studies aimed at elucidating the mechanisms of alternative splicing.

CD117+ small cell lung cancer lacks the asp 816→val point mutation in exon 17

Aims : To determine the frequency of point mutation in c‐kit in CD117+ small cell lung cancer (SCLC). A significant proportion of SCLCs have been documented to be CD117+, thereby signifying they express the c‐kit gene product. This finding suggests this tumour may be a potential target for tyrosine kinase inhibitor (TKI) agents directed at c‐kit. A point mutation in exon 17 of the c‐kit gene, however, can abrogate the binding of TKIs. This being the case, immunohistochemistry is necessary to identify potential candidates for treatment with TKIs, but DNA sequence analysis may need to be performed to determine if these tumours will respond.

Epithelioid angiomyolipoma: Appearance on fine-needle aspiration report of a case

Epithelioid angiomyolipoma is a recently recognized clinicopathologic entity first described by Martignoni et al. in 1995. Since then, several articles have further clarified its histogenesis and histologic features. Due to the presence of polygonal cells with voluminous cytoplasms, this neoplasm is often mistaken for renal‐cell carcinoma. In this case presentation, we describe the cytologic features of an epithelioid angiomyolipoma obtained by fine‐needle aspiration. The histogenesis and how it relates to diagnosis is briefly discussed. The importance of ancillary techniques in the differential diagnosis of epithelioid cells obtained in a renal aspirate is reviewed. Diagn. Cytopathol. 2000;23:192–195.

Breast carcinoma with amplified HER2: a gene expression signature specific for trastuzumab resistance and poor prognosis

Recent trials have shown remarkable efficacy from combined trastuzumab and chemotherapy in the adjuvant setting of breast cancer. In spite of these successes, refractory breast cancer has emerged as a clinically problematic outcome for a subset of patients managed this way. In an effort to clarify and optimize the treatment regimens for breast cancer patients who are candidates to receive trastuzumab, we sought to analyze whether a distinctive genetic signature could be characterized that would reliably predict the treatment outcome. The ability to predict who will respond and who will become refractory to this agent will allow for improved, rational clinical management of these patients and further stratify the personalized nature of this treatment regimen. In this study, 41 consecutive cases of breast carcinoma with well-documented amplification of the human epidermal growth factor receptor-2 gene and corresponding banked fresh-frozen tissue were identified and divided into two separate groups based on whether they received trastuzumab or not. The first group consisted of 12 patients who had received trastuzumab in the adjuvant setting, of which three later experienced tumor recurrence. The second group consisted of 10 patients not treated with trastuzumab, of which 6 were later found to have recurrence. Differentially expressed genetic profiles were determined using human genome-wide Illumina Bead Microarrays. The differentially expressed genes for non-recurrence vs recurrence in the trastuzumab-treated group were distinct from those in the same comparison group in the untreated group. Differential expression of key genes indentified in this study might offer an insight into a possible mechanism of trastuzumab resistance in breast carcinoma, and may emerge as potential predictive biomarkers indicative of trastuzumab resistance.

Manual Exfoliation Plus Immunomagnetic Bead Separation as an Initial Step Toward Translational Research

CONTEXT The development of biotechnologic platforms capable of high throughput analysis has ushered in a promising new era of translational medicine. However, most studies to date are based on in vitro cell lines or substitute models for human disease. Although these model systems have proven insightful, it is readily becoming apparent that human clinical tissue must be studied in order to fully understand all the nuances of human disease. Studies that are based on human tissue, however, are limited by qualitative and quantitative issues, factors often precluding their use in high throughput studies. OBJECTIVE To develop a simple and rapid tissue procurement protocol for use in obtaining a homogeneous epithelial cell population from clinical tissue and the recovery of nucleic acids and proteins of high quality and quantity. Also, to determine if the technique preserves tissue, thereby allowing morphologic correlation with molecular findings. DESIGN Performance of manual exfoliation to procure cells from clinical resection specimens and use of immunomagnetic beads embedded with the antibody ber-Ep4 for the positive enrichment of a homogeneous epithelial cell population. Nucleic acids and proteins are then separated using a phenol plus guanidine thiocyante solution. Nucleic acids and proteins are quantitated and qualitatively analyzed using standard laboratory techniques. RESULTS Nucleic acids and proteins of high quality and quantity were recovered following manual exfoliation and immunomagnetic bead separation. Tissue architecture was not destroyed, thus permitting histologic and molecular correlation. CONCLUSIONS A simple and reproducible protocol is presented that may enable the molecular profiling of clinically resected tissue. Although the technique is currently limited to certain tissue and tumor types, further research will broaden its overall application.

An exfoliation and enrichment strategy results in improved transcriptional profiles when compared to matched formalin fixed samples

BackgroundIdentifying the influence formalin fixation has on RNA integrity and recovery from clinical tissue specimens is integral to determining the utility of using archival tissue blocks in future molecular studies. For clinical material, the current gold standard is unfixed tissue that has been snap frozen. Fixed and frozen tissue however, both require laser capture microdissection to select for a specific cell population to study. The recent development of a sampling method capable of obtaining a viable, enriched cell population represents an alternative option in procuring cells from clinical material for molecular research purposes. The expression profiles of cells obtained by using this procurement approach, in conjunction with the profiles from cells laser capture microdissected from frozen tissue sections, were compared to the expression profiles from formalin fixed cells to determine the influence fixation has on expression profiles in clinical material.MethodsTriplicate samples of non-neoplastic colonic epithelial cells were recovered from a hemicolectomy specimen using three different procurement methods from the same originating site: 1) an exfoliation and enrichment strategy 2) laser capture microdissection from formalin fixed tissue and 3) laser capture microdissection from frozen tissue. Parameters currently in use to assess RNA integrity were utilized to assess the quality of recovered RNA. Additionally, an expression microarray was performed on each sample to assess the influence each procurement technique had on RNA recovery and degradation.ResultsThe exfoliation/enrichment strategy was quantitatively and qualitatively superior to tissue that was formalin fixed. Fixation negatively influenced the expression profile of the formalin fixed group compared to both the frozen and exfoliated/enrichment groups.ConclusionThe exfoliation/enrichment technique represents a superior alternative in tissue procurement and RNA recovery relative to formalin fixed tissue. None of the deleterious effects associated with formalin fixation are encountered in the exfoliated/enriched samples because of the absence of its use in this protocol. The exfoliation/enrichment technique also represents an economical alternative that will yield comparable results to cells enriched by laser capture microdissection from frozen tissue sections.

Presence of the BRAF V600E point mutation in morphologically benign appearing thyroid inclusions of cervical lymph nodes

The biologic nature of morphologically bland-appearing thyroid inclusions in cervical lymph nodes continues to be a controversial topic. The diagnosis of benignity entails a much more conservative clinical approach than does malignancy. Arriving at the correct interpretation, however, can be difficult when only morphologic examination is performed. Incorporating the use of the recently identified BRAF V600E point mutation, a highly specific biomarker for papillary carcinoma of the thyroid, may provide a useful adjunct in assessing the biologic nature of morphologically bland-appearing thyroid inclusions in cervical lymph nodes. In this case report, bland thyroid inclusions were noted in addition to a primary papillary carcinoma of the thyroid and morphologically recognizable cervical lymph node metastasis. Cells from these separate entities were procured by lasercapture microdissection, and the DNA was isolated, amplified, and sequenced. Molecular analhsis provided integral data indicating these morphologically bland thyroid inclusions were malignant, findings not readily apparent by morphologic examination alone.

Conditional Prerequisites for Microchannel Cytologic Analysis on Wet Mount (Fluid-Based) Biopsies

Advanced capabilities in genomic sequencing developed in the research sector will soon enter the clinical arena. Issues such as the proportioning of patient specimen material for traditional bright‐field microscopic evaluation or dedication for molecular analysis will intensify, particularly in situations of small core biopsies. Microfluidics appears aptly suited as a platform capable of allowing traditional cytologic diagnostics and downstream molecular analysis from the same specimen. However, clarification is needed to determine that forces which act on cells in a fluidic environment do not drastically alter their cytologic features.