A Historical Perspective and Modern Techniques in Pulmonary Pathology
Moderators: Dr. Henry D. Tazelaar, Dr. Ming S. Tsao and Dr. Brendan Mullen
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 
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
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
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 . 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 .
3. What are the Main Types of Microdissection?
Manual microdissection and laser-capture
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
, 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
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
lung carcinomas ,
environmental lung diseases
pulmonary vascular diseases
obstructive pulmonarydisease, asthma and airway remodeling
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
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
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.
- Shibata T, et al., Genetic classification of lung adenocarcinoma based on array-based comparative genomic hybridization analysis: its association with clinicopathologic features, Clin Cancer Res. 2005 Sep 1;11(17):6177-85.
- Sasatomi E et al., Genetic profile of cumulative mutational damage associated with early pulmonary adenocarcinoma: bronchioloalveolar carcinoma vs. stage I invasive adenocarcinoma, Am J Surg Pathol. 2004 Oct;28(10):1280-8.
- Dacic S et al., Loss of heterozygosity patterns of sclerosing hemangioma of the lung and bronchioloalveolar carcinoma indicate a similar molecular pathogenesis, Arch Pathol Lab Med. 2004 Aug;128(8):880-4. Review
- Keohavong P et al., Detection of K-ras and p53 mutations in sputum samples of lung cancer patients using laser capture microdissection microscope and mutation analysis, Anal Biochem. 2004 Jan 1;324(1):92-9
- Murase T et al., Clonality analysis of different histological components in combined small cell and non-small cell carcinoma of the lung, Hum Pathol. 2003 Nov;34(11):1178-84.
- Miura K et al., Laser capture microdissection and microarray expression analysis of lung adenocarcinoma reveals tobacco smoking- and prognosis-related molecular profiles, Cancer Res. 2002 Jun 1;62(11):3244-50.
- Ratschiller D et al., Cyclin D1 overexpression in bronchial epithelia of patients with lung cancer is associated with smoking and predicts survival, J Clin Oncol. 2003 Jun 1;21(11):2085-93.
- Dacic S et al., Molecular pathogenesis of pulmonary carcinosarcoma as determined by microdissection-based allelotyping, Am J Surg Pathol. 2002 Apr;26(4):510-6
- Wistuba II et al., Molecular genetics of small cell lung carcinoma, Semin Oncol. 2001 Apr;28(2 Suppl 4):3-13. Review
- Hoang CD et al., Analysis of paired primary lung and lymph node tumor cells: a model of metastatic potential by multiple genetic programs, Cancer Detect Prev. 2005;29(6):509-17. Epub 2005 Nov 10
- Hoang CD et al., Expression profiling of non-small cell lung carcinoma identifies metastatic genotypes based on lymph node tumor burden, J Thorac Cardiovasc Surg. 2004 May;127(5):1332-41; discussion 1342.
- Fernando HC et al., Comparison of mutational changes in involved N1 lymph nodes with those in primary tumors in stage II non-small cell lung cancer: a pilot study, J Thorac Cardiovasc Surg. 2004 Jan;127(1):87-91
- Sasatomi E et al. , Comparison of accumulated allele loss between primary tumor and lymph node metastasis in stage II non-small cell lung carcinoma: implications for the timing of lymph node metastasis and prognostic value, Cancer Res. 2002 May 1;62(9):2681-9.
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- Keohavong P et al., Detection of p53 and K-ras mutations in sputum of individuals exposed to smoky coal emissions in Xuan Wei County, China, Carcinogenesis. 2005 Feb;26(2):303-8. Epub 2004 Nov 25.
- Roberts E et al., Application of laser capture microdissection and protein microarray technologies in the molecular analysis of airway injury following pollution particle exposure, J Toxicol Environ Health A2004 Jun 11;67(11):851-61
- Hunt JD et al., Differences in KRAS mutation spectrum in lung cancer cases between African Americans and Caucasians after occupational or environmental exposure to known carcinogens, Cancer Epidemiol Biomarkers Prev. 2002 Nov;11(11):1405-12
- Taatjes DJ et al., Laser-based microscopic approaches: application to cell signaling in environmental lung disease, Biotechniques. 2001 Oct;31(4):880-2, 884, 886-8, 890, 892-4. Review
- Kelly MM et al., Cell Specific Gene Expression in Patients with Usual Interstitial Pneumonia, Am J Respir Crit Care Med. 2006 May 25
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- Brocker V et al., Fibroblasts of Recipient Origin Contribute to Bronchiolitis Obliterans in Human Lung Transplants, Am J Respir Crit Care Med. 2006 Mar 9
- Ionescu DN, Hunt JL, Lomago D, Yousem SA., Recurrent sarcoidosis in lung transplant allografts: granulomas are of recipient origin, Diagn Mol Pathol. 2005 Sep;14(3):140-5
- Kleeberger W et al., Increased chimerism of bronchial and alveolar epithelium in human lung allografts undergoing chronic injury, Am J Pathol. 2003 May;162(5):1487-94
- Selva E et al., The value of polymerase chain reaction detection of Mycobacterium tuberculosis in granulomas isolated by laser capture microdissection, Pathology. 2004 Feb;36(1):77-81
- Zhu G et al., Gene expression in the tuberculous granuloma: analysis by laser capture microdissection and real-time PCR, Cell Microbiol. 2003 Jul;5(7):445-53
- Kwapiszewska G et al., Expression profiling of laser-microdissected intrapulmonary arteries in hypoxia-induced pulmonary hypertension, Respir Res. 2005 Sep 19;6:109
- Machado RD et al., Investigation of second genetic hits at the BMPR2 locus as a modulator of disease progression in familial pulmonary arterial hypertension, Circulation. 2005 Feb 8;111(5):607-13
- van der Strate BW et al., Cigarette Smoke-induced Emphysema: A Role for the B Cell?, Am J Respir Crit Care Med. 2006 Apr 1;173(7):751-8. Epub 2006 Jan 6
- Kelly MM et al., Epithelial expression of profibrotic mediators in a model of allergen-induced airway remodeling, Am J Respir Cell Mol Biol. 2005 Feb;32(2):99-107. Epub 2004 Nov 24
- Fuke S et al., Chemokines in bronchiolar epithelium in the development of chronic obstructive pulmonary disease, Am J Respir Cell Mol Biol. 2004 Oct;31(4):405-12. Epub 2004 Jun 25
- Kikuchi T et al., Expression profiles of non-small cell lung cancers on cDNA microarrays: identification of genes for prediction of lymph-node metastasis and sensitivity to anti-cancer drugs, Oncogene. 2003 Apr 10;22(14):2192-205.
- Rosell R et al., Mutations in the tyrosine kinase domain of the EGFR gene associated with gefitinib response in non-small-cell lung cancer, Lung Cancer. 2005 Oct;50(1):25-33
- Valencia JC et al., Tissue-Specific Renin-Angiotensin System in Pulmonary Lymphangioleiomyomatosis (LAM), Am J Respir Cell Mol Biol. 2006 Feb 10
- Dacic S et al.,Genotypic analysis of pulmonary Langerhans cell histiocytosis, Hum Pathol. 2003 Dec;34(12):1345-9
- Yousem SA et al., Pulmonary Langerhans' cell histiocytosis: molecular analysis of clonality., Am J Surg Pathol. 2001 May;25(5):630-6.
- Ionescu DN et al., Pulmonary meningothelial-like nodules: a genotypic comparison with meningiomas, Am J Surg Pathol. 2004 Feb;28(2):207-14
- O'Briain DS et al., Sorting out mix-ups: the provenance of tissue sections may be confirmed by PCR using microsatellite markers. Am J Clin Pathol 1996;106: 758-764.
- Hunt JL et al., A microdissection and molecular genotyping assay to confirm the identity of tissue floaters in paraffin-embedded tissue blocks. Arch Pathol Lab Med 2003;128:213-217