Laser Capture Microscopy Research Articles

Abstract B2-57: Genomic analysis of metachronous pancreatic ductal adenocarcinoma

Abstract Background: Improving outcomes for pancreatic ductal adenocarcinoma (PDAC) requires more complete understanding of pancreatic carcinogenesis, including tumor heterogeneity and mutational evolution. Such studies in PDAC are hampered by presentation at advanced stages and rarely operable recurrences. We present a case of metachronous PDAC managed by serial resections and interrogated by whole genome sequencing of all three lesions. Methods: Tumors underwent either laser capture microscopy or flow cytometry of fresh frozen tissue. Germline DNA was obtained from normal tissue. Genomic DNA was isolated, and sequencing libraries were prepared. Whole genome sequencing was performed on the Illumina HiSeq2500 system. Pipeline analysis included quality control, alignment to the reference genome (hg19), variant calling and annotation. Structural variants were called by the union of two tools, DELLY and CREST. Copy number variants were called using an in-house tool. Single nucleotide polymorphisms and short insertions and deletions were identified by the intersection of two tools, Strelka and MuTect, and annotated using ANNOVAR. All structural, copy number and non-silent variants were manually reviewed in the Integrated Genome Viewer. Secondary analyses were done in R. Representative sections of the three tumors also underwent formalin fixation, paraffin embedding, sectioning at five micron thickness and hematoxylin and eosin staining for microscopic analysis. Results: The first tumor was located in the head of the pancreas and resected by Whipple operation, the second and third in the body and tail, respectively, and resected by completion pancreatectomy 14 months later. The patient received chemotherapy in the interim. Histologically, all 3 tumors were moderately differentiated adenocarcinomas with focal mucinous areas and extensive lymphovascular and perineural invasion. There were no distinguishing morphological features. For the 3 lesions, we achieved average sequence coverage of 52.3x, 54.3x, and 70.7x, respectively. A total of 69 structural variants and 62 copy number variants were identified, with concordance of 75% and 92% in all three lesions. A total of 61 non-silent exonic variants were identified, with concordance of 33% in all three lesions. A KRAS (NM_004985) c.G34C missense and TP53 (NM_001126115) c.G418A missense were present in all three, whereas a SMAD4 (NM_005359) c.118delC frameshift was present in only the second and third tumors. Hierarchical clustering by non-silent variants, copy number variants, and an intersect of all variants showed the tumors in the head and tail to be more closely related to one another than to the body lesion, whereas clustering by structural variants showed little difference between the three. Conclusions: The highly similar structural and copy number profiles across the three lesions suggests that these tumors arose from a common precursor, and that large chromosomal changes occurred early in its mutational evolution. The poor concordance of simple somatic mutations imply that these evolved gradually and are responsible for tumor heterogeneity and possibly acquired phenotypes, include therapeutic resistance. This study also demonstrates that isolated, intra-parenchymal metastases (as opposed to de novo tumors within a “field defect”) are possible with PDAC, which has been suggested in case reports but never definitively proven, further implying that these patients may benefit from re-operation despite disease relapse. Further analysis will include mathematical modeling of mutation rates and validation in three additional metachronous cases identified in our biospecimen repository and in specimens obtained by our rapid autopsy program. Citation Format: Ashton A. Connor, Michelle Chan-Seng-Yue, Robert E. Denroche, Ayelet Borgida, Sheng-Ben Liang, Faiyaz Notta, Lincoln Stein, Michael H. Roehrl, John McPherson, Steven Gallinger. Genomic analysis of metachronous pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr B2-57.

Abstract A1-17: Genomic analysis of metachronous pancreatic ductal adenocarcinoma

Abstract Background: Improving outcomes for pancreatic ductal adenocarcinoma (PDAC) requires more complete understanding of pancreatic carcinogenesis, including tumor heterogeneity and mutational evolution. Such studies in PDAC are hampered by presentation at advanced stages and rarely operable recurrences. We present a case of metachronous PDAC managed by serial resections and interrogated by whole genome sequencing of all three lesions. Methods: Tumors underwent either laser capture microscopy or flow cytometry of fresh frozen tissue. Germline DNA was obtained from normal tissue. Genomic DNA was isolated, and sequencing libraries were prepared. Whole genome sequencing was performed on the Illumina HiSeq2500 system. Pipeline analysis included quality control, alignment to the reference genome (hg19), variant calling and annotation. Structural variants were called by the union of two tools, DELLY and CREST. Copy number variants were called using an in-house tool. Single nucleotide polymorphisms and short insertions and deletions were identified by the intersection of two tools, Strelka and MuTect, and annotated using ANNOVAR. All structural, copy number and non-silent variants were manually reviewed in the Integrated Genome Viewer. Secondary analyses were done in R. Representative sections of the three tumors also underwent formalin fixation, paraffin embedding, sectioning at five micron thickness and hematoxylin and eosin staining for microscopic analysis. Results: The first tumor was located in the head of the pancreas and resected by Whipple operation, the second and third in the body and tail, respectively, and resected by completion pancreatectomy 14 months later. The patient received chemotherapy in the interim. Histologically, all 3 tumors were moderately differentiated adenocarcinomas with focal mucinous areas and extensive lymphovascular and perineural invasion. There were no distinguishing morphological features. For the 3 lesions, we achieved average sequence coverage of 52.3x, 54.3x, and 70.7x, respectively. A total of 69 structural variants and 62 copy number variants were identified, with concordance of 75% and 92% in all three lesions. A total of 61 non-silent exonic variants were identified, with concordance of 33% in all three lesions. A KRAS (NM_004985) c.G34C missense and TP53 (NM_001126115) c.G418A missense were present in all three, whereas a SMAD4 (NM_005359) c.118delC frameshift was present in only the second and third tumors. Hierarchical clustering by non-silent variants, copy number variants, and an intersect of all variants showed the tumors in the head and tail to be more closely related to one another than to the body lesion, whereas clustering by structural variants showed little difference between the three. Conclusions: The highly similar structural and copy number profiles across the three lesions suggests that these tumors arose from a common precursor, and that large chromosomal changes occurred early in its mutational evolution. The poor concordance of simple somatic mutations imply that these evolved gradually and are responsible for tumor heterogeneity and possibly acquired phenotypes, include therapeutic resistance. This study also demonstrates that isolated, intra-parenchymal metastases (as opposed to de novo tumors within a “field defect”) are possible with PDAC, which has been suggested in case reports but never definitively proven, further implying that these patients may benefit from re-operation despite disease relapse. Further analysis will include mathematical modeling of mutation rates and validation in three additional metachronous cases identified in our biospecimen repository and in specimens obtained by our rapid autopsy program. Citation Format: Ashton A. Connor, Michelle Chan-Seng-Yue, Robert E. Denroche, Ayelet Borgida, Sheng-Ben Liang, Faiyaz Notta, Lincoln Stein, Michael H. Roehrl, John McPherson, Steven Gallinger. Genomic analysis of metachronous pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-17.

Lace plant ethylene receptors, AmERS1a and AmERS1c, regulate ethylene-induced programmed cell death during leaf morphogenesis.

The lace plant, Aponogeton madagascariensis, is an aquatic monocot that forms perforations in its leaves as part of normal leaf development. Perforation formation occurs through developmentally regulated programmed cell death (PCD). The molecular basis of PCD regulation in the lace plant is unknown, however ethylene has been shown to play a significant role. In this study, we examined the role of ethylene receptors during perforation formation. We isolated three lace plant ethylene receptors AmERS1a, AmERS1b and AmERS1c. Using quantitative PCR, we examined their transcript levels at seven stages of leaf development. Through laser-capture microscopy, transcript levels were also determined in cells undergoing PCD and cells not undergoing PCD (NPCD cells). AmERS1a transcript levels were significantly lower in window stage leaves (in which perforation formation and PCD are occurring) as compared to all other leaf developmental stages. AmERS1a and AmERS1c (the most abundant among the three receptors) had the highest transcript levels in mature stage leaves, where PCD is not occurring. Their transcript levels decreased significantly during senescence-associated PCD. AmERS1c had significantly higher transcript levels in NPCD compared to PCD cells. Despite being significantly low in window stage leaves, AmERS1a transcripts were not differentially expressed between PCD and NPCD cells. The results suggested that ethylene receptors negatively regulate ethylene-controlled PCD in the lace plant. A combination of ethylene and receptor levels determines cell fate during perforation formation and leaf senescence. A new model for ethylene emission and receptor expression during lace plant perforation formation and senescence is proposed.

Abstract 2080: Differential transcription profile of epithelia in fimbria versus the ampulla of the fallopian tube

Abstract Background: Recently described precursors of high-grade serous carcinoma (HGSC), the p53 signature, a latent precursor, and Serous Tubal Intraepithelial Carcinoma (STIC), a pre-malignant precursor, occur most frequently at the distal and fimbriated end of the fallopian tube (FTE). In 3 previous reports, we have demonstrated that the FTE of BRCA1 mutation carriers, at genetic risk of HGSC, have altered signaling pathways compared to controls. Ovarian production of ROS is released after the LH surge to induce ovulation. Reactive oxygen species (ROS) have been implicated in serous carcinogenesis. The objective of this study is to compare the transcriptome profiles of normal fimbria (high-risk epithelia prone to transformation) FTE and normal ampulla (low-risk epithelia) FTE which may lead to understanding the distal end of the fallopian tube as the preferential anatomic location of the fallopian to tube for cellular transformation. Method: Snap-frozen matched fimbria and ampulla tissues were controlled for age and ovarian cycle status. Cases included 12 luteal phase and 12 follicular phase women at no known risk for ovarian cancer. Laser capture microscopy was used to microdissect FTE cells, using 7-10 sections per case. Total RNA was isolated, RNA extracted and cDNA amplified. The expression profiles were generated using Affymetrix Human Genome HTA-2.0 Array. Results: Using gene level differential expression analysis with the Affymetrix Expression Console software, we performed unsupervised hierarchical clustering analysis with all 24 samples. We used a fold change of < -2 or > 2 and ANOVA p-value < 0.05 as a cut-off criteria for selecting genes. The cases clustered predominantly by ovarian cycle status rather than by their differences in anatomical origin or their matched pair. There were 427 genes differentially expressed amongst the 4 groups– Fim-Luteal, Fim-Follicular, FT-Luteal and FT-Follicular. Independent of ovarian cycle status, very few differences (35 genes– SALL1, ME1– higher in the ampulla; GSTA1, GSTA2, SERPINA3-higher in the fimbria– genes involved in metabolic pathways) were observed between the ampulla and fimbria FTE. Conclusions: The epithelia of the anatomically high-risk fallopian tube– the fimbria, show few differences in gene expression profiles compared to the lower risk portion– the ampulla. Expression differences predominantly are in response to the hormonal milieu, post-ovulation with an antioxidant program The increased anatomic risk of the fimbria is likely due to effects of the microenvironment, such as repeated exposure to follicular fluid at ovulation (higher ROS) and antioxidant genes and their interaction with DNA repair genes (like TP53), rather than intrinsic differences of the FTE in the two sites. Citation Format: Sophia H. George, Anca Milea, Noor Hera Salman, Patricia Ann Shaw. Differential transcription profile of epithelia in fimbria versus the ampulla of the fallopian tube. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2080. doi:10.1158/1538-7445.AM2015-2080

Mechanisms of Mammalian Middle Ear Ossicle Transition from the Reptilian Jaw Joint

During synapsid evolution, postdentary elements in the reptilian jaw transitioned into the middle ear of early mammals. Separation from the dentary allowed unconstrained evolution of the middle ear ossicles, resulting in increased hearing sensitivity and amplified frequency range. Given the importance of such an innovation, it is essential to understand the evolutionary pathway that led to the current phenotype, as this has implications for understanding the development of all mammals. Using an extant model system, Monodelphis domestica, we investigate the developmental basis of the definitive mammalian middle ear. We utilize micro‐CT imaging to characterize the morphological events underpinning the transition, cryosectioning and immunohistochemistry to identify cellular processes behind the morphological events, and laser capture microscopy followed by RNA Sequencing to identify changes in gene expression driving the cellular processes. Among our findings are: decreasing size and rearward movement of the middle ear ossicles appear to be false illusions created by the continued growth and expansion of the surrounding skull elements; separation of Meckel's cartilage from the malleus, occurring at postnatal day 20, is facilitated by apoptosis and is a prerequisite for the onset of hearing; separation and functional changes are due to alterations in timing and expression levels of key genes.Supported by NSF Graduate Research Fellowship and Doctoral Dissertation Improvement Grant.

Attributing Oncogenic Human Papillomavirus Genotypes to High-grade Cervical Neoplasia

Human papillomavirus (HPV) is found in most women with high-grade cervical intraepithelial neoplasia (CIN) 2/3 in cervical cytology and biopsies. Multiple high-risk HPV (hrHPV) genotypes are present in 15% to 50% of cytology samples. We have shown by laser-capture microscopy (LCM)-polymerase chain reaction (PCR) that each lesion is associated with a single hrHPV type. Attribution of hrHPV types to CIN2/3 is important to understand the oncogenic role of different types and the limitations of cytologic typing. We studied hrHPV genotypes in 257 women with histologic CIN2/3 referred on the basis of abnormal cytology. HPV typing was done on cytology and CIN2/3 biopsies. If the whole-tissue section of the biopsy was positive for multiple hrHPV types, LCM-PCR was performed. We found 181 (70%) single and 71 (28%) multiple hrHPV infections in cytology, with 5 (2%) cases HPV-positive only on whole-tissue section PCR. Of cases with multiple cytologic hrHPV infections, 47/71 (66%) showed a single type in CIN2/3 lesions. In total, in 232 of 257 (90%) women with CIN2/3, a single hrHPV type caused CIN2/3. One was nonattributable on the LCM level. The remaining 24 women had 2 or more contiguous or separated lesions, each associated with a single hrHPV infection. The probability of HPV16 being present in CIN2/3, if detected in cytology, was 0.96 (95% confidence interval=0.90-0.98). LCM-PCR confirms that only 9% of histologic CIN2/3 is associated with multiple hrHPV types, much less than cytology would indicate, and each lesion was associated with a single hrHPV infection.

Cell type-specific affinity purification of nuclei for chromatin profiling in whole animals.

Analyzing cell differentiation during development in a complex organism requires the analysis of expression and chromatin profiles in individual cell types. Our laboratory has developed a simple and generally applicable strategy to purify specific cell types from whole organisms for simultaneous analysis of chromatin and expression. The method, termed INTACT for Isolation of Nuclei TAgged in specific Cell Types, depends on the expression of an affinity-tagged nuclear envelope protein in the cell type of interest. These nuclei can be affinity-purified from the total pool of nuclei and used as a source for RNA and chromatin. The method serves as a simple and scalable alternative to FACS sorting or laser capture microscopy to circumvent the need for expensive equipment and specialized skills. This chapter provides detailed protocols for the cell-type specific purification of nuclei from Caenorhabditis elegans.

A novel microdissection approach to recovering mycobacterium tuberculosis specific transcripts from formalin fixed paraffin embedded lung granulomas.

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most common microdissection technique used today. In this technique, a laser is used to focally melt a thermoplastic membrane that overlies a dehydrated tissue section(1). The tissue section composite is then lifted and separated from the membrane. Although this technique can be used successfully for tissue examination, it is time consuming and expensive. Furthermore, the successful completion of procedures using this technique requires the use of a laser, thus limiting its use. A new more affordable and practical microdissection approach called mesodissection is a possible solution to the pitfalls of LCM. This technique employs the MESO-1/MeSectr system to mill the desired tissue from a slide mounted tissue sample while concurrently dispensing and aspirating fluid to recover the desired tissue sample into a consumable mill bit. Before the dissection process begins, the user aligns the formalin fixed paraffin embedded (FFPE) slide with a hematoxylin and eosin stained (H&E) reference slide. Thereafter, the operator annotates the desired dissection area and proceeds to dissect the appropriate segment. The program generates an archived image of the dissection. The main advantage of mesodissection is the short duration needed to dissect a slide, taking an average of ten minutes from set up to sample generation in this experiment. Additionally, the system is significantly more cost effective and user friendly. A slight disadvantage is that it is not as precise as laser capture microscopy. In this article we demonstrate how mesodissection can be used to extract RNA from slides from FFPE granulomas caused by Mycobacterium tuberculosis (Mtb).

Proteomic analysis of laser capture microscopy purified myotendinous junction regions from muscle sections.

The myotendinous junction is a specialized structure of the muscle fibre enriched in mechanosensing complexes, including costameric proteins and core elements of the z-disc. Here, laser capture microdissection was applied to purify membrane regions from the myotendinous junctions of mouse skeletal muscles, which were then processed for proteomic analysis. Sarcolemma sections from the longitudinal axis of the muscle fibre were used as control for the specificity of the junctional preparation. Gene ontology term analysis of the combined lists indicated a statistically significant enrichment in membrane-associated proteins. The myotendinous junction preparation contained previously uncharacterized proteins, a number of z-disc costameric ligands (e.g., actinins, capZ, αB cristallin, filamin C, cypher, calsarcin, desmin, FHL1, telethonin, nebulin, titin and an enigma-like protein) and other proposed players of sarcomeric stretch sensing and signalling, such as myotilin and the three myomesin homologs. A subset were confirmed by immunofluorescence analysis as enriched at the myotendinous junction, suggesting that laser capture microdissection from muscle sections is a valid approach to identify novel myotendinous junction players potentially involved in mechanotransduction pathways.

Altered choroid plexus gene expression in major depressive disorder.

Given the emergent interest in biomarkers for mood disorders, we assessed gene expression in the choroid plexus (CP), the region that produces cerebrospinal fluid (CSF), in individuals with major depressive disorder (MDD). Genes that are expressed in the CP can be secreted into the CSF and may be potential biomarker candidates. Given that we have previously shown that fibroblast growth factor family members are differentially expressed in post-mortem brain of subjects with MDD and the CP is a known source of growth factors in the brain, we posed the question whether growth factor dysregulation would be found in the CP of subjects with MDD. We performed laser capture microscopy of the CP at the level of the hippocampus in subjects with MDD and psychiatrically normal controls. We then extracted, amplified, labeled, and hybridized the cRNA to Illumina BeadChips to assess gene expression. In controls, the most highly abundant known transcript was transthyretin. Moreover, half of the 14 most highly expressed transcripts in controls encode ribosomal proteins. Using BeadStudio software, we identified 169 transcripts differentially expressed (p < 0.05) between control and MDD samples. Using pathway analysis we noted that the top network altered in subjects with MDD included multiple members of the transforming growth factor-beta (TGFβ) pathway. Quantitative real-time PCR (qRT-PCR) confirmed downregulation of several transcripts that interact with the extracellular matrix in subjects with MDD. These results suggest that there may be an altered cytoskeleton in the CP in MDD subjects that may lead to a disrupted blood-CSF-brain barrier.

Oncogenic Human Papillomavirus–infected Immature Metaplastic Cells and Cervical Neoplasia

Persistent cervical high-risk human papillomavirus (HR-HPV) infection results in high-grade cervical intraepithelial neoplasia (CIN2/3) and cervical carcinoma. The susceptibility of the cervix to HPV carcinogenesis and the importance of HPV18 in cervical carcinoma despite relative infrequency in CIN2/3 could be linked to HR-HPV infection of immature metaplasia (IM) at the squamocolumnar junction. Atypical IM (AIM) is an equivocal category used to describe changes in IM suggestive of high-grade neoplasia, which causes diagnostic and management problems. We used laser capture microscopy combined with polymerase chain reaction in 24 women with HPV18, HPV16, or other HPV infections on cytologic analysis and a cervical loop electrosurgical excision procedure to locate HR-HPV in cervical tissue. HPV18-positive AIM and CIN2/3 were present in 7/12 cases with HPV18 on cytologic analysis. In 2 cases with HPV18 and other HPV types, HPV18 was only present in AIM and not in CIN2/3. HPV16-positive AIM was present in 3/7 and HPV16-positive CIN2/3 in 5/7 cases with HPV16. No cases had HPV16 AIM without CIN2/3. Other HR-HPV-positive AIM and CIN2/3 cases were present, respectively, in 1/6 and 5/6 cases positive for HR-HPV types other than HPV16/18. In a subset, 94% HPV18 AIM regions showed CK17 and p16 positivity, and 41% were CK7 positive. CIN2/3 and AIM with other HR-HPVs showed similar patterns. AIM was a particular feature of HPV18 infection in women with CIN2/3. HR-HPV infection of CK7/17-positive AIM expressing p16 was particularly seen for HPV18 with and without classical CIN2/3 and should be regarded as a high-grade precancer.

Fluorescence-based laser capture microscopy technology facilitates identification of critical in vivo cytomegalovirus transcriptional programs.

Cytomegalovirus gene expression in highly permissive, cultured fibroblasts occurs in three kinetic classes known as immediate early, early, and late. Infection of these cells results in a predictable transcriptional program leading to high levels of virus production. Infection of other, so-called, nonpermissive cell types results in a transcriptional program that either fails to produce virus particles or production is substantially reduced compared to fibroblasts. We have found that CMV gene expression profiles in tissues from infected hosts differ greatly from those observed in infected tissue culture cells. The number of viral genes expressed in tissues is much more limited, and the number of highly active genes does not correlate with viral DNA load. Additionally, viral gene expression in vivo is tissue selective with no two tissues expressing the exact same viral gene profile. Thus, in vivo CMV gene expression appears to be governed by mechanisms that are still uncharacterized. Cytomegalovirus remains in a persistent phase for the lifetime of the host. During this phase only a limited number of host cells are infected, and it is very difficult to detect CMV gene expression in whole tissues without sub-fractionating infected vs. uninfected cells. Herein, we describe the development of a fluorescence-based laser capture microscopy technique coupled with small sample size microarray analysis to determine the viral gene expression in 50-100 infected cells isolated from frozen RCMV-infected tissue sections.

Transcriptional regulation by nicotine in dopaminergic neurons

Dopaminergic neurons in the substantia nigra pars compacta (SNc) degenerate in Parkinson's disease. These neurons robustly express several nicotinic acetylcholine receptor (nAChR) subtypes. Smoking appears to be neuroprotective for Parkinson's disease but the mechanism is unknown. To determine whether chronic nicotine-induced changes in gene expression contribute to the neuroprotective effects of smoking, we develop methods to measure the effect of prolonged nicotine exposure on the SNc neuronal transcriptome in an unbiased manner. Twenty neurons were collected using laser-capture microscopy and transcriptional changes were assessed using RNA deep sequencing. These results are the first whole-transcriptome analyses of chronic nicotine treatment in SNc neurons. Overall, 129 genes were significantly regulated: 67 upregulated, 62 downregulated. Nicotine-induced relief of endoplasmic reticulum (ER) stress has been postulated as a potential mechanism for the neuroprotective effects of smoking. Chronic nicotine did not significantly affect the expression of ER stress-related genes, nor of dopamine-related or nAChR genes, but it did modulate expression of 129 genes that could be relevant to the neuroprotective effects of smoking, including genes involved in (1) the ubiquitin–proteasome pathway, (2) cell cycle regulation, (3) chromatin modification, and (4) DNA binding and RNA regulation. We also report preliminary transcriptome data for single-cell dopaminergic and GABAergic neurons isolated from midbrain cultures. These novel techniques will facilitate advances in understanding the mechanisms taking place at the cellular level and may have applications elsewhere in the fields of neuroscience and molecular biology. The results give an emerging picture of the role of nicotine on the SNc and on dopaminergic neurons.

Proteomic anatomy of human skin

The extracellular matrix is composed of a variety of proteins which are essential for growth, wound healing, and fibrosis. It provides both structural support as well as contributing to the regulation of the local microenvironment. To further characterize the molecular composition of human skin we have undertaken a proteomic approach to identify proteins in three skin regions from two locations. Using laser microdissection, extracellular matrix was obtained from three distinct regions (basement membrane, papillary dermis, and reticular dermis) of formalin-fixed, paraffin embedded tissue from normal human leg and breast skin. The proteome of these regions was determined by mass spectrometric analysis. This study provides a relative quantitative assessment, including protein composition and relative abundance, of the proteins found in different skin regions giving rise to a "proteomic anatomy" of skin. Our findings indicate that there was little difference detected in the subproteomes of the dermal and papillary regions and little difference between the proteomes of leg skin compared to breast skin. One finding of interest is the identification of tenascin-X only in the breast dermis and serum amyloid P-component in the leg dermis. The results of this proteomic analysis corroborate much of the information on the protein composition identified by other methodologies found in the literature but provide additional insight as to localization of skin proteins in the various regions of skin. One potential outcome of this study is that identification of a more global proteomic composition in normal skin may serve as the basis for characterizing and comparing the skin proteomes from a variety of disease states, which may lead to a more complete understanding of the pathology of the disease as well as new therapeutic treatments. This investigation underscores the power of proteomics to bring semiquantitative, non-presumptive molecular characterization to the field of histological anatomy. Traditionally, anatomy relied on visual or histochemical structural characterization which generally involved some level of understanding of the area of interest. With the advent of laser microdissection or laser capture microscopy localization of anatomical structures of interest can be correlated to molecular composition by virtue of mass spectrometric determination of the proteome of that structure. One potential outcome of this study is that identification of a more global proteomic composition in normal skin may serve as the basis for characterizing and comparing the skin proteomes from a variety of disease states, which may lead to a more complete understanding of the pathology of the disease as well as new therapeutic treatments.

Insights from transcriptional profiling the mouse hindbrain: Novel feedback between retinoids and Hox genes

Adult vertebrate hindbrain regulates critical body functions such as sleep, respiration and heart rate. During embryogenesis hindbrain forms seven transient swellings called rhombomeres, each with unique morphology and gene expression patterns. Rhombomere specification and regulation is controlled by transcription factors including Hox family and Krox20. Disruption of these genes changes the number and properties of rhombomeres and leads to neuronal disorganization causing animal lethality.We are using transcriptional profiling and functional validation to reveal rhombomeric‐specific patterns of gene expression regulated by these key transcription factors. We isolated individual rhombomeres from 9.5 dpc mouse embryos using laser capture microscopy and performed transcriptional profiling on either single rhombomeres from the wild type hindbrains or whole hindbrains of wild type and Hoxa1, Hoxa2, Hoxb1 and Krox20 mutants. Analyses and validation of these results have uncovered several novel targets and pathways associated with hindbrain specification. For example, Hox genes which are induced by retinoids in turn modulate retinoid metabolism. These feedback loops are important for hindbrain patterning.Grant Funding Source: Stowers Institute for Medical Research

Mass spectrometry imaging: linking molecule profiles to tissue spatial distribution

Advances in Bioanalytical Mass Spectrometry: 5th Symposium of the Göttingen Proteomics ForumMass Spectrometry with Spatial Resolution: MALDI-Imaging and Laser Capture MicroscopyGöttingen, Germany, 22 November 2012MALDI mass spectrometry imaging (MSI) combines the speed and molecular specificity of MALDI-MS detection with information on spatial organization. In the last years, MSI found large application in proteomics research for determining the spatial distribution of compounds in biological tissues and started to draw increasing interest in clinical research. To shed light on the new developments in the field of MSI, the Göttingen Proteomics Forum organized a symposium that was held in Göttingen as part of the series of regular symposia organized by the members of the Göttingen Proteomics Forum. The symposium was on 22 November 2012, with more than 80 delegates that attended the event entitled ‘Mass spectrometry with spatial resolution: MALDI-imaging and laser capture microscopy’. The one-day agenda consisted of nine oral presentations from renowned experts in the field with subsequent discussion sessions. As usual, the meeting was fruitful and offered a good platform for discussion between the delegates and proteomics specialists.

Comparative study of epidermal growth factor receptor mutation analysis on cytology smears and surgical pathology specimens from primary and metastatic lung carcinomas

The detection of epidermal growth factor receptor (EGFR) mutations on small biopsy or fine-needle aspiration samples is required to guide therapy in nonsmall cell lung cancer (NSCLC). In this study, the authors compared results from EGFR mutation testing on both cytologic smears and surgical specimens and also compared the performance of platforms using 2 different technologies (pyrosequencing and real-time polymerase chain reaction) for both specimen types. Specimens from 114 patients were divided into 2 subsets. The first subset had 60 paired cytology smears and surgical specimens, including 37 paired specimens from the same site and 23 paired specimens from different sites. The second subset consisted of nonpaired cytology smears and formalin-fixed, paraffin-embedded (FFPE) tissues (including 8 cell blocks), which were compared on the pyrosequencing and real-time polymerase chain reaction platforms. Laser-capture microscopy was used to enrich tumor in the FFPE specimens before DNA extraction. All cytology smears that were used in the study were adequate for analysis on both platforms. Comparison between smears and concurrent FFPE tissues from the same anatomic site had a concordance rate of 97%. The concordance rate between the pyrosequencing platform and the real-time polymerase chain reaction platform was 84% and 85% for FFPE tissues and cytology smears, respectively. The current results indicated that direct extraction and analysis of EGFR mutations from cytology smears can be performed successfully on both a pyrosequencing platform and a real-time polymerase chain reaction platform with results comparable to those achieved in matched surgical specimens. In fine-needle aspiration/endobronchial ultrasound samples with limited tissue, cytology smears can be important for molecular analysis. Cancer (Cancer Cytopathol) 2013;121:361-369. © 2012 American Cancer Society.

RNA localization in the honeybee (Apis mellifera) oocyte reveals insights about the evolution of RNA localization mechanisms

Subcellular localization of RNAs is a critical biological process for generation of cellular asymmetries for many cell types and a critical step in axis determination during the early development of animals. We have identified transcripts localized to the anterior and posterior of honeybee oocyte using laser capture microscopy and microarray analysis. Analysis of orthologous transcripts in Drosophila indicates that many do not show a conserved pattern of localization. By microinjecting fluorescently labeled honeybee transcripts into Drosophila egg chambers we show that these RNAs become localized in a similar manner to their localization in honeybee oocytes, indicating conservation of the localization machinery. Thus while the mechanisms for localizing RNA are conserved, the complement of localized RNAs are not. We propose that this complement of localized RNAs may change relatively rapidly through the loss or evolution of signal sequences detected by the conserved localization machinery, and show this has occurred in one transcript that is localized in a novel way in the honeybee. Our proposal, that the acquisition of novel RNA localization is relatively easy to evolve, has implications for the evolution of symmetry breaking mechanisms that trigger axis formation and development in animal embryos.

E4 Antibodies Facilitate Detection and Type-Assignment of Active HPV Infection in Cervical Disease

High-risk human papillomavirus (HPV) infections are the cause of nearly all cases of cervical cancer. Although the detection of HPV DNA has proved useful in cervical diagnosis, it does not necessarily predict disease presence or severity, and cannot conclusively identify the causative type when multiple HPVs are present. Such limitations may be addressed using complementary approaches such as cytology, laser capture microscopy, and/or the use of infection biomarkers. One such infection biomarker is the HPV E4 protein, which is expressed at high level in cells that are supporting (or have supported) viral genome amplification. Its distribution in lesions has suggested a role in disease staging. Here we have examined whether type-specific E4 antibodies may also allow the identification and/or confirmation of causal HPV-type. To do this, type-specific polyclonal and monoclonal antibodies against three E4 proteins (HPV-16, -18, and -58) were generated and validated by ELISA and western blotting, and by immunohistochemistry (IHC) staining of epithelial rafts containing these individual HPV types. Type-specific detection of HPV and its associated disease was subsequently examined using formalin-fixed paraffin-embedded cervical intra-epithelial neoplasias (CIN, (n = 247)) and normal controls (n = 28). All koilocytotic CIN1 lesions showed type-specific E4 expression of their respective HPV types. Differences were noted amongst E4 expression patterns in CIN3. HPV-18 E4 was not detected in any of the 6 HPV-18 DNA-positive CIN3 lesions examined, whereas in HPV-16 and -58 CIN3, 28/37 (76%) and 5/9 (55.6%) expressed E4 respectively, usually in regions of epithelial differentiation. Our results demonstrate that type-specific E4 antibodies can be used to help establish causality, as may be required when multiple HPV types are detected. The unique characteristics of the E4 biomarker suggest a role in diagnosis and patient management particularly when used in combination.

An induction of microRNA, miR-7 through estrogen treatment in breast carcinoma

BackgroundEstrogen plays an important role in the development of estrogen-dependent breast carcinoma. Recently, several studies demonstrated a possible involvement of several micro RNAs (miRNAs) in the development of resistance to endocrine therapy in breast cancer patients, but the correlation between estrogen actions and miRNA expression in breast carcinoma still remains largely unknown. Therefore, in this study, we examined the in vitro effects of estrogen upon miRNA expression profiles in breast carcinoma.MethodsWe first screened the miRNA expression profiles induced by 17β-Estradiol (E2) using RT2 miRNA PCR Array in the ER-positive breast carcinoma cell line MCF-7. We identified miR-7 as the important miRNA associated with estrogen actions in these cells and further examined the changes of estrogen-dependent EGFR expression by miR-7 in ER-positive or -negative breast carcinoma cell lines including MCF-7. We also evaluated the correlation between miR-7 and EGFR expression in breast carcinoma cells derived from 21 patients using laser capture microdissection combined with quantitative reverse transcriptase-PCR.ResultsSeventeen miRNAs were significantly induced by E2 treatment in the MCF-7 cell line. Among 17 miRNAs induced by estradiol treatment, only miR-7 expression was significantly decreased by subsequent ICI treatment. The expression of miR-7 was up-regulated 2.94-fold by E2 treatment. miR-7 was reported to suppress epidermal growth factor receptor (EGFR) expression in several human malignancies. Transfection of miR-7 significantly suppressed EGFR mRNA levels in MCF-7 cells. Depletion of E2 from cell culture media also increased the expression level of EGFR mRNA in MCF-7 and T-47D cells but not in ER-negative, MDA-MB-231 and SK-BR-3 cells. We also evaluated the status of miR-7 in breast carcinoma tissues, but the correlation between the status of miR-7 and EGFR in carcinoma cells isolated by laser capture microscopy was not detected.ConclusionsThese results suggest that miR-7 may play a role in the development of resistance to endocrine therapy in breast cancer patients through regulating EGFR expression of carcinoma cells.