CRC FISH Validation Abstract Accepted for Poster Presentation

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					CRC Tissue Core FISH Studies
Accepted for Poster Presentation ASH 2008
Validation of CLL FISH Panel Scoring by Members of
the Chronic Lymphocytic Leukemia Research
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Stephanie A. Smoley , Daniel L. Van Dyke , Neil E. Kay , Nyla A. Heerema , Marie L. dell' Aquila ,
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Prasad Koduru , Paola Dal Cin , Laura Rassenti , John C. Byrd , Kanti R. Rai , Jennifer R.
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Brown , Andrew W Greaves , Thomas J. Kipps and Gordon W. Dewald , (1)Mayo Clinic, Stabile 6-
28, 200 First Street, Rochester, MN 55905-0001, (2)Mayo Clinic, 200 First Street SW, Rochester, MN
55905, (3)Hematology/Oncology division, Mayo Clinic, 200 First St., SW, Rochester, MN 55905, (4)Ohio
State University, 129 Hamilton Hall, 1645 Neil Avenue, Columbus, OH 43210, (5)University of California
San Diego, San Diego, CA 92093, (6)North Shore Hospital, 300 Community Drive, Manhasset, NY
11030, (7)Brigham & Women's Hospital, Boston, MA 02115, (8)Medicine, Moores UCSD Cancer Center,
3855 Health Sciences Dr., Mail Code 0820, La Jolla, CA 92093-0820, (9)The Ohio State University, B302
Starling Loving Hall, 320 West 10th Ave., Columbus, OH 43210, (10)Long Island Jewish Med. Ctr., 270-
06 76th Ave., New Hyde Park, NY 11040, (11)Dana-Farber Cancer Inst., 44 Binney St., Boston, MA
02115, (12)Univ. of California, San Diego, 3855 Health Science Dr., #0820, La Jolla, CA 92093-0820,
(13)Mayo Clinic, 200 First Ave. SW, Hilton 970, Rochester, MN 55905

Fluorescence in situ hybridization (FISH) probes and analysis methods for B-cell Chronic
Lymphocytic Leukemia (CLL) vary extensively among cytogenetic laboratories. This is not
unexpected, as neither national nor international standards have been established for most FISH
studies. Lack of standardization is problematic when data collected at multiple institutions are
used for clinical correlative studies. To circumvent such problems, the five participating
laboratories in the CLL Research Consortium (CRC) designed and executed a joint CLL FISH
validation study. Methods: Initially a survey was sent to assess equipment, methods and
experience with FISH for CLL. In a pilot study to compare laboratory performance in scoring
patient samples, slides from ten patients were prepared and sent to each participating lab to be
hybridized with five probe sets (= 50 hybridizations) and analyzed according to their local
protocol. In a second pilot study, slides from two patient samples and identical probe sets were
sent to the participating labs where hybridization and analysis were carried out according to their
local protocol. Next, technologists and directors from all participating labs attended a workshop
where technologists working in pairs scored nuclei together, techniques and scoring criteria were
established, and consensus reached on other concerns. In a proficiency test nine months after the
workshop, slides from two patient samples (10 hybridizations) were hybridized and scored
according to each lab’s protocol and results shared using a common reporting form. Results:
Survey results indicated that four labs used the same commercially available CLL FISH panel,
and one used a combination of probes from the same vendor plus several home-brew probes.
Each lab scored between 100 and 200 nuclei per hybridization site, and each independently set
normal cutoff values. The FISH panel included probes to detect 11q, 13q, and 17p deletions,
trisomy 12, and IGH gene rearrangement. One lab included probes to detect 6q deletion. In the
first pilot study each lab used their hybridization methods, probe sets, and scoring criteria.
Differences among labs were observed due to variations in probe strategy, reporting of
anomalies, and perhaps most important, scoring criteria. Probe strategy differences resulted in
variable reporting of 11q- vs monosomy 11 and 12q duplication vs trisomy 12. Some
participants reported 13q-x1 and 13q-x2 as subclones and some reported only 13q-. One lab
reported an IGH rearrangement whereas the others scored IGH as normal. In the second pilot
study each lab used the same methods and probe sets to facilitate comparison of scoring by the
technologists. All labs correctly identified the abnormalities, and there were no false positive
results. Minor scoring differences were attributed to variation in scoring criteria or inexperience
with an unfamiliar FISH probe strategy. The proficiency test that followed the workshop
demonstrated 100% concordance in identification of abnormalities. Inter-lab scoring was much
improved compared to the first pilot study. The only exceptions were a 13q- range of 72-90% in
one case, and a 17p- range of 38-67% in another case. Conclusion: The pilot studies identified a
need to develop common scoring criteria. The subsequent workshop and proficiency test
demonstrated that the collaborative effort resulted in more standardized scoring among the CRC
laboratories. Our collaborative study emphasizes the need to establish rigorous standards and
guidelines for FISH procedures and scoring criteria. Standardization of FISH methods among
participating laboratories will enhance the confidence in FISH studies for both clinical
applications and cooperative intergroup clinical research.

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