FISH Scoring for CLL: Comparison of Methods That Assess Round Versus Non-Round Nuclei,
Authors: Greipp Patricia T. , Smoley Stephanie A. , Kay Neil , Zent Clive S. , Eckel-Passow Jeannette E. , Sharma Ruchi G. , Dyke Daniel L. Van 
Published: October 2019

Abstract 3538

Background:

Cytogenetics detection of common recurring genetic defects by FISH is an important clinical prognostication tool in CLL and is frequently used by clinicians for counseling and risk stratification. Thus, any improvement in FISH technology is a welcome advance in our care for CLL patients. Mayo Clinic CLL FISH analyses are prepared directly from buffy coat samples rather than mononuclear cells or lymphocytes. FISH probes (deletion 6q, 11q, 13q, 17p, +12 & IGH/CCND1) are scored among consecutive nuclei without selection for nuclear morphology (so called clinical scoring). CLL B-cells are observed, microscopically, primarily as round lymphocytes with round nuclei, and we found (ASH 2010, #3606) that the % abnormal nuclei in the CLL round cell population is significantly higher than that of the non-round nuclei population. In 79 of 87 (91%) patients, the % abnormal nuclei was greater in round vs unselected nuclei (mean difference 24%; range 1.5 – 57%). Round nuclei (p = 0.004) correlated better with % B-cells by flow cytometry than either consecutive unselected nuclei (p = 0.046) or the lobed (non-round) nuclei (p = 0.40). Utilizing this “Poor Man's” sorting technique, we proposed that scoring consecutive round nuclei would improve sensitivity of detection of CLL FISH defects in patients, especially those with low B-cell counts. In a retrospective study of Mayo Clinic patients with normal baseline FISH who had a FISH defect in follow-up, we showed that by re-scoring 28 baseline cases for round nuclei exclusively, 12 originally normal samples remained normal but 16 (57%) were abnormal using the “Poor Man's” scoring technique. In the present study, we prospectively compared FISH scoring on unsorted samples using the “Poor Man's” technique vs the clinical technique.

Methods:

After IRB approval, we collected 63 consecutive unsorted Mayo Clinic patient blood samples referred for CLL FISH testing with an absolute lymphocyte count (ALC)< 30K × 109/L, and scored each sample using both the “Poor Man's” round nuclei method and the standard clinical method. The kappa statistic was used to adjust for chance agreement. Bland Altman plots of difference (“Poor Man's” result minus clinical result) vs the average of the two methods were generated.

Results:

Median age of the 40 males (64%) and 23 females (36%) was 66 years (range 36–87), and median ALC was 6.803 (range 0.980–29.750). The most common FISH defects were 11q-, trisomy 12 & 13q- by clinical technique (14%, 22% & 46% respectively) as well as by “Poor Man's” method (16%, 24% & 49% respectively). A high degree of agreement was observed between the two methods when positive/negative comparisons were made (p<0.0001). Additional FISH defects were observed in 12 of 63 (19%) patients (one 11q-, one +12; six 13q-, and four 17p-) when we utilized the “Poor Man's” method that were not observed by the clinical method.

Discussion:

The “Poor Man's” sorting method compares well with the clinical method for the presence and absence of FISH defects. The abnormal CLL FISH patterns are truly confined to round nuclei. The “Poor Man's” method is more sensitive than the clinical method, resulting in higher reported abnormal cells and detection of more defects. Importantly, additional FISH defects were identified in 12 patients via “Poor Man's” method that were not observed by clinical technique and 6 of these involved a change in the patients' hierarchical (Döhner) classification due to detection of the more unfavorable prognostic markers of trisomy 12 or deletion 11q or 17p. Thus, selectively scoring FISH in the round nuclei increases the sensitivity for detection of a genetic defect in CLL and provides a practical framework for prognostication in CLL patients with low lymphocyte counts and, we would speculate, including those in clinical remission or with minimal residual disease.

Disclosures:

Kay: Biothera: Research Funding; Clegene: Research Funding; Cephalon: Research Funding; Genentech: Research Funding; Glaxo Smith Kline: Research Funding; Hospira: Research Funding; Novartis: Research Funding; Supergen: Research Funding; Calistoga: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Emergent Biosolutions (Formerly Trubion): Membership on an entity's Board of Directors or advisory committees.