I’m sure you will agree our first Expert Access Live-on-Line of Spring, “Laser Capture Microdissection and Its Applications to Genomics & Proteomics” was outstanding. Our host Dr. James Wittliff is currently on-line and awaiting your questions, so please submit them now.

We would like to extend our most sincere thanks to the Bayer Corporation for making this program possible.

when will we see this new generation of molecular tests on the biopsy tissue be seen en masse?
Washington, DC

James L. Wittliff, Ph.D., M.D.:As I mentioned in the answer to another question, this new generation of molecular tests on tissue biopsies is rapidly being applied to a variety of diseases. It is my personal opinion that it will first be applied in the management of cancer, particularly breast and prostate, where significant strides have been made. Furthermore, new studies such as those I described in this presentation indicate that gene expression profiling of LCM procured breast cancer cells is ready for use in a national cooperative clinical trial such as those conducted by the National Surgical Adjuvant Breast & Bowel Project (NSABP) or the Southwest Oncology Group (SWOG). My view and that of others in this field is that there will be clinical tests of both gene and protein expression profiling of LCM procured cells within 2-3 years.

Dr. Wittliff, thank you for your excellent presentation on such an interesting topic. In the present health care environment where Histology staffing is not always adequate to meet the usual demands of the laboratory, how can a lab reliably meet the turn-around-time required for LCM?
Fallbrook, California

James L. Wittliff, Ph.D., M.D.: LCM technology is highly standardized and easily taught to laboratory staff with an intermediate background in histology. I have found that laboratory staff involved in the use of the LCM system and its application in basic and clinical research are highly motivated by this new approach. It is visible, interesting and relevant to many avenues of investigation. Several individuals with whom I have worked have indicated that it provides a new challenge for their skills and stimulates their interest in learning its application in more sophisticated areas of genomics and proteomics. The training sessions that I indicated in the answer to one of the other questions is rapidly advancing the skill levels of laboratory personnel. Turn around time for preparing a frozen tissue sections for LCM and procurement of cells on three individual caps requires 1-2 hours, including extraction of macromolecules. I believe that other laboratory staff will be involved in the final stages of the clinical test such as RNA amplification and microarray or protein separation by 2-D gel electrophoresis and analysis. The problems of staff training and turn around time are being met with a new enthusiasm because of this emerging technology.

Do you know how many facilities perform LCM? What is the greatest challenge to offering this technology?
Andover, MA

James L. Wittliff, Ph.D., M.D.:3) Currently more than 500 institutions worldwide have established facilities for LCM use in both basic and clinical research. Among the greatest challenges for offering the LCM technology as a clinical chemistry tool are 1) training of personnel, 2) establishment of proficiency surveys and 3) physician education of utility. Regarding training, currently the laboratories of Drs. Lance Liotta and Bob Bonner at the National Cancer Institute offer a 2-3 day training session at the NIH, on a monthly basis at no charge. Contact Dr. Lance Liotta at his email address ( lance@helix.nih.gov ) to schedule a session, at which you may bring and prepare selected tissues from your institution. Arcturus Engineering, Inc. offers comprehensive training for a fee at their Mountain View, CA location. Contact them through their website ( www.arctur.com ). Regarding interlaboratory quality control, my laboratory and others who have worked with the AACC and CAP are exploring various tissues, fixation and staining conditions, etc. to establish a proficiency survey similar to that developed for estrogen and progestin receptors used by the cooperative clinical trial groups (e.g., NSABP, SWOG) and the College of American Pathologists. Physician understanding of the clinical applications of LCM technology in disease diagnosis, assessment of prognosis and therapy response is growing, but I believe it will require the use of LCM in either gene or protein expression profiling in several national clinical trials before we will make that “leap” to a routine clinical test.

Can you offer an estimate of what the cost per test is to perform laser capture microdissection in a hospital setting?
Augusta, GA

James L. Wittliff, Ph.D., M.D.: The cost per test with LCM will greatly depend upon the type analyses, i.e., genomic or proteomic, single analyte, etc., to be performed with the population of cells captured. The industry and application will have to mature more before I can provide an estimate of the cost.

Dr. Wittliff, other than those you describe, do you foresee any other applications of laser capture microdissection?
Stamford, CT

James L. Wittliff, Ph.D., M.D.:The literature is replete with new and exciting applications of LCM to human disease. I only have time to mention a few very recent applications. These include molecular analysis of liver stage parasites (Sacci et al., Mol Biochem Parasitol 119:285, 2002), LOH of uterine cervix and adenocarcinoma in situ (Acevedo et al., Cancer 94:793, 2002), atherosclerotic lesions (Trogan et al., Proc Natl Acad Sci USA 99:2234, 2002), ocular inflammation (Foxman et al., J Immunol 168:2483, 2002), central nervous system (Tuaillon & Chan, Curr Mol Med 1:259, 2001), cardiovascular disease (Stagliano et al., Ann NY Acad Sci 947:344, 2001). Again, the advantage of LCM technology is that you may remove individual populations of various types of cells (intact) in close proximity to each other. Clearly, there is no limit to the application in both basic and clinical research.

How soon will this method be used in general cancer management?
Madisonville LA

James L. Wittliff, Ph.D., M.D.: To date, there are more than 30 clinical studies indicating the utility of either genomic or proteomic based studies of LCM procured cells. Perhaps one of the greatest advantages of LCM technology to our understanding of disease, is the ability to separate both normal and neoplastic cells from the same tissue biopsy. By performing either gene or protein expression profiles of the two types of cells, one obtains a more appropriate comparison of cellular status, since the normal cells serve as the internal control. For those of you interested, you can either search PubMed or contact me by email and I will send a list of references that appear clinically relevant. My personal assessment of the field is that either genomic or proteomic studies of LCM procured cells will be used within 2-3 years in management of selected cancers such as those of the breast and prostate.

Dear Dr. Wittliff: How many tests do you need to perform a month to make this a viable in house offering?
New Orleans

James L. Wittliff, Ph.D., M.D.: I don’t have a direct answer to your question regarding the number of LCM-based tests per month that will be required to make this a viable in-house offering. However, recent investigations clearly show that six biopsies with three independent captures may be processed through LCM and macromolecular extraction in an eight-hour workday. For gene expression analysis, most laboratories pool samples and amplify the RNA in batches of 10-20 samples. For protein expression profiling a similar number of samples are processed as a set. It appears that a “well-oiled” lab could handle 20 individual tissue specimens per week through all steps required of either protein or gene expression analyses. While this does not sound like a large number, keep in mind that the number of analytes examined and the magnitude and potential of the results in clinical management is considerable. It is not unreasonable that these tests will cost $1,500-$2,500 during the early stages of their use in clinical medicine, in my opinion.

Dr. Wittliff, How can Diagnostic Companies best help clinicians and laboratorians best harvest the fruits of laser capture microdissection? What is needed right now or soon to help this technology reach its full potiential for patients?
San Diego, CA

James L. Wittliff, Ph.D., M.D.: 8) Diagnostic companies can best serve patients and clinicians by enhancing educational opportunities describing these new techniques. For example, collection of a viable tissue biopsy is an essential requirement to insure either genomic or proteomic studies of LCM procured cells accurately reflect cellular status. The temperature and other conditions in an operating theatre are not usually adequate for maintaining the biochemical and structural integrity of tissue biopsies. Therefore, there is a serious need to inform both surgeons and pathologists of the importance for maintaining the tissue specimen on ice after excision, handling it in a manner reducing RNase (for gene expression profiling) and protease (for protein expression profiling) exposure. Both surgeons and pathologists were exposed to the fundamentals of this change in tissue handling procedures, when tissue-based markers such as estrogen and progestin receptors were first employed in clinical medicine. Unfortunately, with the decrease in biochemical testing of tissue-based markers and the increase in immunohistochemical testing of formalin-fixed/paraffin embedded tissues, clinicians have paid less attention to conditions necessary for maintaining tissue integrity required for genomic and proteomic analyses. I firmly believe that they will support the use of added care as they gain appreciation of the power and application of these emerging clinical chemical tests. One of the greatest needs to determine the full potential of this technology and approach in clinical medicine is its application in a multi-institutional clinical trial. I am convinced that LCM technology and its application in several genomic and proteomic approaches are well established and ready for implementation in protocols of national cooperative clinical groups. In virtually every example of therapy advancements in the treatment of cancer, results of national cooperative clinical trials such as those conducted by the National Surgical Adjuvant Breast & Bowel Project (NSABP), Cancer and Leukemia Group B (CALGB), Eastern Cooperative Oncology Group (ECOG), Gynecologic Oncology Group (GOG), North Central Cancer Treatment Group (NCCTG) and Southwest Oncology Group (SWOG) have led the way for establishing the use of new clinical tests as well as therapies.

What is the resolution? How certain can you be that a single nucleus is dissected?
Bronx, NY

James L. Wittliff, Ph.D., M.D.: The laser beam may be focused for a spot size of 7.5 to 30 um by adjusting the power and duration settings. Routinely I am able to procure a single, intact carcinoma cell using the spot size of 7.5 um using a power setting of 35-45 mW with a duration setting of 2.5-3.0 msec and a 5 micron tissue section. It is our procedure to stain the tissue section with H & E using a modified technique maintaining macromolecular integrity. This stain easily indicates that you are removing a single cell by observing it on the screen. Of course, we have not been able to perform either gene or protein expression profiles of a single cell.

Does insurance cover the tests?
Burmingham, AL

James L. Wittliff, Ph.D., M.D.: Although Laser Capture Microdissection has been issued a CPT code according to my understanding, I do not know if insurance covers the test.

Does this technology have a forensic application? There are times when mixed cell/tissue sources need to be separated -- mixed suspect-victim, or multiple suspects each of whom may have contributed blood/body fluids?
Milwaukee

James L. Wittliff, Ph.D., M.D.:There is a definite advantage to using LCM procured cells for DNA analysis in the forensic setting. I know of several studies in which LCM is being used experimentally to separate different cell types (identified by using various immunohistochemical stains) with the emphasis on discerning host-foreign cell properties. In my opinion this is one of the most immediate applications of the LCM technology combined with more defined sets of genes or protein analytes. Imagine the power of a procedure allowing DNA analyses of 500 cells.

Thanks for a presentation on something I know very little about. Are the studies you described still considered "research," that is "not covered by Medicare/insurance," or are these considered, at least in some cases, the standard of care -- and reimburseable?
Chicago, Illinois

James L. Wittliff, Ph.D., M.D.: The studies that I have described in my particular presentation are still considered research and not covered by insurance. However, LCM has a CPT code of which I have no details. As mentioned earlier, implementation of studies such as those I have described, in a national cooperative clinical trial would greatly advance our understanding of their application in clinical medicine. Surely, Medicare/Insurance coverage would be forthcoming.

Dear Dr. Wittliff: Could you please comment on how this technology usually managed - in AP or CP. If CP, is it Chemistry?
Houston, TX

James L. Wittliff, Ph.D., M.D.: It is my understanding that the LCM technology combined with emerging tests involving proteomics/genomics would be managed in clinical pathology. It is unclear if the LCM portion, which involves pathologic description of the tissue, would be managed in anatomic pathology while the actual chemistry would be managed in CP. It is my understanding that the College of American Pathologists is discussing this currently.

Dr. Wittliff Do you have a standard type of slide and tissue preparation (freezing/fixation/microtoming/staining) that provides great cell morphology so that a person untrained in histology can find specific cells for acquisition? Training requires the most time and with inconsistent results with fixation etc., it takes even more time than most labs have to spare. Or do you have a good reference that would be of some help? Thank you
Greenfield, Indiana

James L. Wittliff, Ph.D., M.D.: 14) I have written a chapter in a volume of Methods in Enzymology, which is in press, describing details related to this question. As soon as this is published, I’ll be happy to make it available to all participants in this educational on-line program. Briefly, it is necessary to use RNase free conditions prepping the cryostat appropriately before taking tissue sections. Specimens are frozen in Optimum Cutting Temperature compound (TissueTek® OCT medium) sectioned and collected on sterile microscope slides without a cover slip and retained frozen by placing on a flat surface of dry ice to preserve labile macromolecules. We recommend that glass slides without coatings (uncharged) be used to enhance LCM removal of selected cells. Frozen tissue sections on slides are stored in sterile plastic slide holders – 80 degrees C until analyses. Details are contained in the chapter and I am happy to share these with any of the participants, if you email me. Let me emphasize that all of the procedures are now standardized and do not require a great level of skill to obtain reducible results. Note, the training courses offered at the National Cancer Institute or at Arcturus Engineering, Inc. that I mentioned earlier, are useful in getting started.

how many tissue samples do you routinely prepare in parallel and what is a feasible maximum number to handle properly?
Duesseldorf Germany

James L. Wittliff, Ph.D., M.D.: As I mentioned in more detail earlier, an individual comfortable with the LCM procedures may procure cells from six different tissue sections with three independent captures per section, and extract macromolecules in an eight hour work day. The biochemical analyses (genomic or proteomic) require considerably more time and most investigators process these in batches of 10-20 samples for either RNA amplification or protein separation. Analyses of gene and protein expression profiles are evolving and requires sophisticated software. However, a number of laboratories have established this as a routine and are processing dozens of samples per week.

I read with interest your review in the Clinical Ligand Assay Society's journal last year. My question deals with quality control of the system. Is there an accepted strategy for quality control, i.e. to be sure that the cells that are being examined are the correct ones? It seems that morphology will obviously play a central role in assuring quality, but is there any other means that might be used?
Baltimore, MD

James L. Wittliff, Ph.D., M.D.: It appears that quality control must be maintained at several levels. Firstly, the individual utilizing the Laser Capture Microdissection system to remove a particular cell type, e.g., carcinoma cells will have to be educated in their recognition by a pathologist. However, this is not difficult once the individual gains experience. Clearly, at this level there must be over-sight, and the fact that the PixCell II® LCM system has an extensive archiving system in which all images of the tissues before and after capture, as well as cells procured on the caps are stored, insures review by the pathologist. Finally, we and others have demonstrated that a variety of immunohistochemical stains used to identify various cell types, do not interfere with LCM procurement of cells and their subsequent analyses for macromolecules.

Kudos to you and AACC for highlighting this exciting technology, Dr. Wittliff. It would seem that LCM could be a useful tool in gathering homogeneous cellular material for use in clinical trials of potential drug compounds. Do you know of any group that is examining this application?
Bethesda, MD

James L. Wittliff, Ph.D., M.D.: 17) I am not aware that any multi-institutional groups are combining these strategies. I believe this is because the technology has emerged so rapidly that those of us who are employing it in our research have had little opportunity to share the exciting findings with clinical colleagues. During this on-line session this afternoon, I have made several references to the need for employing LCM technology combined with genomic/proteomic approaches. Most of the published studies emphasize the findings of a single group. However, I believe there is enough evidence published to warrant the application of these approaches in a variety of treatment trials of breast, prostate and lung carcinomas. It is my understanding that several of the cooperative clinical trial groups have explored the use of microarrays (gene chips) using entire tissue sections. The problem here is one of cellular heterogeneity resulting in gene expression profiles reflecting various cell types. Now that there are sufficient number of laboratories well versed in LCM technology, I would suggest that a multi-institutional drug trial could be conducted whereby carcinoma cells could be collected and extracted by several core laboratories, and the extracts analyzed by microarray or protein chips at other facilities. Very quickly we should gain an understanding of the therapeutic potential of new agents.

Are there any LCM systems which permit faster and higher sample throughput? (I heard that there is one which allows selection of cell groupings for simultaneous capture and catapulting.)
Seattle, WA

James L. Wittliff, Ph.D., M.D.: If I understand the question correctly, there are several different systems available for “capturing” cells. Each of these employs a somewhat different technology. The new volume # 356 (Laser Capture Microscopy and Microdissection, edited by P. Michael Conn) of Methods in Enzymology, Academic Press, contains chapters by a number of investigators with experience in the different instruments/technologies used to remove cells from heterogeneous tissue sections. I am familiar with the AutoPix® LCM, an automated instrument made by Arcturus, which allows the operator to define cellular regions of a tissue section and initiate the rapid collection of cell groups without operator involvement. The advantage here is that the technology permits the collection of numerous geometrically difficult cell groups and structures with minimal contamination by surrounding, unwanted cell types.

Is Arcturus the only company that makes this technology? Do they have a patent? Are other companies interested in marketing the item. It does not seem very well know. Perhaps marketing through one of the large dianostics would be of help?
Detroit

James L. Wittliff, Ph.D., M.D.: As I indicated in the slide presentation, the concept of Laser Capture Microdissection originated with Drs. Lance Liotta, Bob Bonner and Michael Emmert-Buck at the National Cancer Institute of the NIH. Through a CRADA agreement, Arcturus Engineering, Inc. developed this technology culminating in a commercial instrument in 1997. They hold the patent on the PixCell® II & Iie instruments. I believe they are the only company that markets this instrument and their website is www.arctur.com. There are several different systems available for “capturing” cells and cellular structures employing somewhat different technologies. The new volume # 356 (Laser Capture Microscopy and Microdissection, edited by P. Michael Conn) of Methods in Enzymology, Academic Press, contains chapters by investigators describing the different instruments/technologies used to remove cells from heterogeneous tissue sections. I am familiar with the AutoPix® LCM, an automated instrument made by Arcturus, which allows the operator to define cellular regions of a tissue section and initiate the rapid collection of cell groups without operator involvement. The advantage here is that the technology permits the collection of numerous geometrically difficult cell groups and structures with minimal contamination by surrounding, unwanted cell types. One of the principal efforts possible by the large diagnostic companies would be to play a role in comprehensive clinical trials of the LCM technology and its application in genomics and proteomics of human disease.

Thank you for taking time from your hectic schedule to join us for this fascinating session. This Expert session and all previous sessions, are archived on our website and serve as a continuing source of education.

Mark your calendar for our May Expert session, " Mycobacterial Cell Manipulation from Specimen to DNA". Our expert for this program is Dr. Robert-A. Ollar, Director, Molecular Biology Unit, Department of Neurology at St. Vincent’s Hospital and Medical Center, NY. Dr. Ollar will be available on-line May 7 between 1 and 2 pm Eastern to answer your questions. Don’t miss this fascinating topic.

Again, thank you to the Bayer Corporation for making this educational program possible.

See you in May!