Section 6 -

Applications of Microdissection Techniques in Pulmonary Pathology Diana N. Ionescu

Molecular biological testing and its applications in anatomical pathology are becoming more diverse and complex as our understanding of molecular events associated with diseases is growing exponentially. First developed and used in translational research, molecular techniques are becoming more and more part of the diagnostic and prognostic workup of clinical cases. If in the past, a tissue sample was giving us important, but limited information, the amount of information we are able to obtain from the same tissue sample today through the use of molecular techniques is infinitely larger. 1. What is Microdissection? A technique that manually or with the use of commercially available instruments separates tissues or cells of interest. Tissue microdissection is potentially one of the most useful techniques in molecular pathology [1] that separates relatively pure cellular samples of morphologically confirmed cell types in frozen or paraffin-embedded tissues. This technique allows direct visualization and manual selection of areas of interest or individual cells of interest, and their separation from the adjacent tissue components and background material. 2. What is the Rationale for Performing Microdissection? To obtain pure tissue samples or isolated cell populations from which DNA or mRNA will be extracted and serve as the interrogation material for various and complex qualitative and quantitative molecular techniques. Histologically verified tissues are available in many surgical pathology laboratory archives in the form of fresh, or more often, archived paraffin-embedded material and represent an attractive resource for investigators in pathology and basic sciences. The inherent heterogeneity of primary tissues with an admixture of various cell populations can affect the outcome and interpretation of molecular studies. Tissue samples without microdissection may not be ideal for these types of applications because they may be contaminated with normal stromal and lymphoid tissues [2]. Newly developed, highly sophisticated and focused molecular techniques require interogation of selected, pure cell populations for the study of genetic alterations and isolation of genes and proteins. Various molecular techniques including mutation analysis, loss of heterozigosity (LOH), comparative genomic hybridization (CGH), genomic fingerprinting, reverse transcriptase polymerase chain reaction (RT-PCR), differential display and microarray hybridization can be applied to the DNA and RNA extracted from microdissected tissue [4]. 3. What are the Main Types of Microdissection? Manual microdissection and laser-capture microdissection (LCM) Early microdissection techniques involved manual or micromanipulator guidance of a needle or thin blade to isolate the cells of interest under direct microscopic visualization. LCM is a technique recently developed at the National Cancer Institute, which permits the reliable and rapid procurement of smaller cell populations or single cells from tissue sections, in one step. Other modified techniques for microdissection have also been reported [1], such as the use of a hydraulic micromanipulator and starch adhesive gum fractionation, etc., but proved to be less efficient, time consuming and labor intensive and lost popularity. 4. What are the Advantages/Disadvantages of Microdissection? Advantages: Manual microdissection is a fairly simple, quick technique, which does not require expensive equipment or extensive training of the operator. LCM is more precise and obtains nearly pure cell populations and minuscule clusters of cells or single cells. Disadvantages: With manual microdissection it is often difficult to obtain a pure cell population and the technique is dependent on the operator's skills and dexterity. LCM requires special tissue preparation, expensive technology, extensive training of the operator and is labor intensive; it may be also challenging to obtain adequate tissue adherence and extract DNA from the LCM microdissected sample. 5. What are the Limitations of Microdissection? Microdissection requires familiarity with the morphology of the tissue of interest and therefore needs to be performed by pathologists or trained histotechnologists. This technique is also limited by the tissue fixation (i.e. formalin vs. fixative with low pH or containing heavy metals), tissue cross-contamination during processing, type of staining (i.e type of hematoxylin used, pretreatment used for histochemistry or immunohistochemistry), most of which interfere with subsequent DNA or RNA extraction. 6. What are the Main Applications of Microdissection in Pulmonary Pathology? The applications of microdissection in pulmonary pathology, as in other areas of surgical pathology, can be classified into those related to research, quality assurance and diagnosis. A comprehensive list of references on the most recent studies (2000-2006) using microdissection, in various applications in pulmonary pathology, is appended to this handout. 7. What are Some Current Research Applications of Microdissection in Pulmonary Pathology? Research applications in pulmonary include the study of normal and premalignant lesions, lung cancer [pathogenesis [4, 5, 6, 7, 8, 9, 10, 11, 12, 13], metastasis [14, 15, 16, 17, 35], synchronous lung carcinomas [18], targeted therapy [35, 36]] , environmental lung diseases [19, 20, 21, 22], interstitial lung diseases [23, 24], lung transplantation, [25, 26, 27], pulmonary vascular diseases [30, 31], chronic obstructive pulmonarydisease, asthma and airway remodeling [32, 33, 34] and miscellaneous lesions [37, 38, 39, 40]. Microdissection can be used in normal or premalignant tissues for assessment of clonal development. Lung cancer is the one field in pulmonary pathology where most of research applications of microdissection are seen. Molecular assays performed on DNA or RNA extracted from microdissected lung cancers helped with the understanding and analysis of tumor genotype and clonality beyond traditional histopathology. These techniques are still challenged by the intratumoral heterogeneity, as well as theheterogeneity at the gene level of morphologically similar lung cancers. Although these studies continue to bring new insights in the pathogenesis of lung cancer and its metastases, more studies are necessary to correlate tumor genetic profiling with established prognostic factors before we can implement molecular information into the staging and treatment decision. New genomic technologies developed over recent years are able to analyze thousands of genes and their expression profiles simultaneously with the purpose of discovering new cancer biomarkers, improving diagnosis, predicting clinical outcomes of disease and response to treatment, and selecting new targets for novel agents with innovative mechanisms of action. Microdissection of tumor samples was shown to be useful for the study of targeted therapy for lung cancers such as the prediction of tumor sensitivity to anti-cancer drugs and the sensitive detection of EGFR mutation in non-small-cell lung cancer, a strong predictive biomarker for EGFR-targeted protein tyrosine kinase inhibitors. Environmental lung disorders only recently benefited from the application of LCM, as the majority of information regarding the mechanisms by which air pollution particles generates lung injury has been derived from in vitro studies. In 2004 studies demonstrated for the first time the credibility of applying LCM and protein microarray technologies to assess acute lung injury induced by environmental air pollutants. Interstitial lung diseases, some with a dismissal prognosis due to irreversible fibrosis were the subject of studies performed on microdissected lung tissue targets such as fibroblastic foci and helped us in understanding the pathogenesis and potential molecular targets for interfering with fibrogenesis. The combination of microdissection and molecular identity testing proved to be a very elegant approach in studying lung transplantation, particularly in regard to chimerism and donor or recipient cell origin and interaction. Through microdissection ofbronchial and alveolar epithelium, studies have shown increased chimerism during chronic injury in human lung allografts and proved a recipient origin of the histiocytes composing noncaseating granulomas in recurrent sarcoidosis or the fibroblasts involved in the development of bronchiolitis obliterans in the allograft lung. Pulmonary infections and more specifically M. tuberculosis, as well as the kinetics of host gene expressionin granulomas isolated by LCM were detected and studied using real time PCR.LCM and array profiling have revealed several new genes involved in pulmonary vascular diseases andlung vascular remodelling in response to hypoxia. This new approach allows a deeper insight into hypoxic regulatory pathways, specifically in the vascular compartment of the lung.By combining microdissection with reverse transcriptase-polymerase chain reaction and cDNA microarrays, several studies evaluated the role of bronchiolar epithelium as the source of increased inflammatory chemokine levels involved in the development of COPD, asthma and airway remodeling and also demonstrated the potential use of LCM in investigating functional profiles of individual structural and inflammatory cells in human lungs. Genotypic analysis and studies of histogenesis and cell of origin for miscellaneous pulmonary entities such as meningothelial-like nodules, sclerosing hemangioma and pulmonary Langerhans' cell histiocytosis were also performed using microdissection. 8. What are Some Quality Assurance Applications of Microdissection in Pulmonary Pathology? As in any other area of surgical pathology, microdissection can be used in conjunction with molecular identity testing for tissue origin confirmation [1, 2]. In characterization of tissue floaters (tissue carryover artifacts), microdissection is essential for isolating the fragments of the suspected floater from the rest of the tissue sample [41, 42]. 9. What are Some Diagnostic Applications of Microdissection in Pulmonary Pathology? In the last years,PCR performed on granulomas isolated by LCM has proven to be a sensitive, specific and rapid method for the detection of M. tuberculosis in formalin-fixed and paraffin-embedded samples, including small lung biopsies. In respect with lung cancer, clinical applications of microdissection are still limited in both their availability and performance. Molecular techniques applied on microdissected tissue are sporadically used by molecular anatomical pathology laboratories of larger academic institutions in distinguishing multiple primary lung carcinomas from intrapulmonary metastases or the origin of metastases in cases unsorted by other ancillary techniques (i.e. lymph node metastases of lung or head and neck origin). 10. What is the Future of Microdissection for the Pulmonary Pathologist? With the advance in our understanding of molecular biology of cancer and the molecular profiling of lung cancers, an increasing number of molecular assays will become clinically useful in characterization and subclassification of lung cancer patients and in detection of infectious organisms in lung tissues. The role of the future pulmonary pathologist will include providing the clinician with increasingly specific molecular prognostic markers for survival, as well as for targeted, individualized therapies. Therefore, familiarity with advantages, disadvantages, multiple applications and important limitations of molecular testing is essential. Tissue microdissection will remain one of the most useful techniques in supplying the genetic material necessary for the performance of complex molecular tests. References (Studies Involving Microdissection in Pulmonary Pathology, 2000-2006) Sirivatanauksorn Y et al., Laser-assisted microdissection: applications in molecular pathology. J Pathol. 1999 Oct;189(2):150-4. Review. Hunt JL, Finkelstein SD., Microdissection techniques for molecular testing in surgical pathology. Arch Pathol Lab Med. 2004 Dec;128(12):1372-8. Review. Fend F, Raffeld M., Laser capture microdissection in pathology, J Clin Pathol. 2000 Sep;53(9):666-72. Review. Nakamura N, et al., Identification of tumor markers and differentiation markers for molecular diagnosis of lung adenocarcinoma, Oncogene. 2006 Feb 20. 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