Stockero, Kimberly J.

Abstract Introduction: A new isodicentric variant of del(20q) was recently described; namely ider(20)(p11.2)del(20)(q11.2q13.3). The frequency and clinical significance of this anomaly are not yet established. This anomaly is usually missed in routine cytogenetic studies and the common commercial FISH probe, D20S108, does not distinguish between del(20q) and ider(20q). The goals of this study were to determine 1) the frequency and hematopathology of patients (pts) with ider(20q) and 2) the best cytogenetic methods to distinguish among various del(20q) anomalies. Methods: We selected 12 pts referred for chromosome studies with -20,+mar to look for evidence of ider(20q). For comparison, we also randomly selected 12 pts with classical del(20q). Up to 10 bone marrow metaphases were studied for each of these 24 pts using FISH with probes for 20pter, JAG1 (20p12), centromere 20, D20S108 (20q12) and 20qter. We also studied 200 interphase nuclei from each pt using D20S108 and 20qter probes. We reviewed cytogenetic results for a consecutive series of pts in our hematological practice between 9/1/05 and 12/31/05 to establish the frequency of ider(20q). Blood and marrow slides were available for microscopic evaluation in 9 of 12 pts. with ider(20q). Results: By metaphase FISH, each of the 12 pts with del(20q) were 20pter+, JAG1+, D20S108- and 20qter+ indicating an interstitial deletion. Each of the 12 pts with ider(20q) were 20pter-, JAG1-, D20S108-- and 20qter++ indicating an isodicentric chromosome with an interstitial del(20q) on either side of the centromere. The ider(20q) is best detected using probe for D20S108 and 20qter. By FISH each pt with ider(20q) had a mixture of cells with del(20q) and ider(20q), though cells with only del(20q) were not usually detected by chromosome studies. One pt had a subclone with two copies of ider(20q). Interphase FISH identified del(20q) and ider(20q) in each specimen. Hematopathology studies of 9 pts with ider(20q) indicated acute myeloid leukemia (AML) with multilineage dysplasia in 1 and myelodysplastic syndrome in 8 (refractory cytopenia with multilineage dysplasia (RCMD) in 3, RCMD with ringed sideroblasts in 3, atypical myelodysplastic/myeloproliferative disease in 1, and refractory anemia with excess blasts-2 (RAEB-2) in 1). Additional chromosome anomalies were detected in the pts with AML with multilineage dysplasia and RAEB-2. Six pts had dysgranulopoiesis in addition to dysmegakaryopoiesis and/or dyserythropoiesis. Among 5,744 pts in our hematological clinical practice screened by interphase FISH, ider(20q) was seen in 8, but 8 of 78 pts with del(20q) also had ider(20q). Discussion: In clinical practice ider(20q) is common in hematological disorders and occurs in 0.14% of all pts and in 10% of pts with del(20q). This figure may underestimate the true frequency of ider(20q) because novel FISH studies with D20S108 and 20qter are required to confirm its presence. When G-banding preparations indicate -20,+mar, then ider(20q) should be suspected. The literature is sparse but pts with ider(20q) appeared to have a more consistent presentation of multilineage dysplasia with additional involvement of the granulocytic series than pts with del(20q). These results suggest that it is helpful to distinguish among various forms of del(20q) in clinical practice using both chromosome and FISH studies with D20S108 and 20qter.

Abstract Background: Results of conventional cytogenetic studies on specimens processed without B-cell mitogens are usually normal in B-CLL: this observation is often attributed to lack of mitotic B-CLL cells. Using metaphase FISH with D13S319, we now have evidence that these apparently normal mitotic B-CLL cells often exhibit submicroscopic interstitial deletions in 13q14. This observation indicates B-CLL cells do divide in unstimulated cultures, but the abnormal metaphases are not detected because the abnormalities are submicroscopic and represent a low percentage of proliferating cells. Methods: We studied 12 patients with B-CLL who had normal metaphases by conventional cytogenetics, but abnormal interphase nuclei by FISH with D13S319. This cohort consisted of 7 females and 5 males who ranged in age from 50 to 87 years. Clinical information was available for 11 patients: Rai stages were 0, I, II, III, or IV for 6, 1, 1, 2, and 1 patients, respectively; 8 patients received treatment and 3 have died. Conventional cytogenetic studies and metaphase FISH were done on bone marrow specimens that were processed by standard 24 and 48-hour unstimulated cultures with Chang Medium BMC. Interphase FISH studies were done on blood or bone marrow (collected within 3 days of specimens for conventional cytogenetic studies) using probes to detect anomalies of chromosomes 6, 11, 12, 13, 14 and 17. For each patient, metaphase FISH was done with commercial probes from Vysis Inc., Downers Grove, IL for D13S319/13q34 using leftover fixed cells from conventional cytogenetics studies. Results: Metaphase FISH results were normal for 3 bone marrow specimens: the remaining 9 specimens had 1.9 to 13.8% metaphases with interstitial deletions of 13q14. One patient had a cytogenetically visible chromosome 13 deletion in 2.2% of metaphases. In the remaining 8 specimens, the size of the abnormal 13 was similar to the normal 13 homolog and other acrocentric chromosomes. By comparison, interphase FISH revealed 13 to 93% nuclei with 13q-x1 or 13q-x2. Seven patients had additonal anomalies in their interphase nuclei; 3 had 11q-, 2 had 17p-, 1 had +12 and 1 had +IGH. Conclusions: This investigation indicates that metaphase FISH detects submicroscopic deletions in chromosome 13 as well as infrequent mitotic B-CLL cells in patients who have normal conventional cytogenetic results. Therefore, metaphase FISH is a novel and useful technique to detect abnormal mitotic cells in B-CLL in cultures without special B-cell mitogens. The prognostic significance of abnormal metaphase FISH results is still under investigation. We speculate that this method will allow for identification of more aggressive proliferating disease even in so-called “favorable” prognostic groups based on interphase FISH results.

Abstract Background: Loss of p53 is detected by fluorescence in situ hybridization (FISH) in interphase nuclei in 8–10% of patients with B-CLL and has been associated with aggressive disease and poor response to therapy. Results of chromosome studies suggest loss of p53 is often due to structural anomalies of chromosome 17 rather than monosomy 17. To characterize chromosome 17 anomalies in B-CLL, we used interphase and metaphase FISH to study a series of patients with this disorder. Methods: We studied 16 patients with B-CLL who had chromosome 17 anomalies detected by conventional chromosome studies or interphase FISH. The study cohort consisted of 8 females and 8 males (age 54 – 73 years). Clinical data was available for 15 patients: Rai stages were I, II, III and IV for 1, 2, 6 and 6 patients, respectively; 14 patients received treatment and 10 have died. Metaphase and interphase FISH studies were performed using left-over fixed bone marrow or peripheral blood cell pellets from conventional cytogenetic studies. To identify recurrent chromosome 17 breakpoint cluster regions, patients were first studied with p53(17p13.1) and D17Z1(centromere17), and then with FISH probes for SMS(17p11.2) and RARA(17q21). For patients retaining the SMS probe site, we did FISH for HNPP(17p11.2–12) and D17Z1(centromere17). A total of 200 interphase nuclei and up to 20 metaphases were analyzed by each probe set. Results: The karyotype was normal for 3 patients and complex involving various abnormalities of chromosome 17 for the remaining 13. All patients showed loss of one p53 probe site with retention of both chromosome 17 centromeres. The SMS locus was absent in 13 patients indicating a breakpoint cluster region between the centromere and SMS probe site. In the remaining 3 patients, HNPP was absent, but the SMS probe site was retained indicating chromosome breakage between the SMS and HNPP probe sites. Metaphase FISH results indicate loss of p53 was due to an isochromosome 17p11.2 in 4 patients and an unbalanced translocation involving 17p11.2 in 12 patients. Conclusions: This study shows that loss of p53 in B-CLL cells is due to isochromosome formation or unbalanced translocations that involve a ~6,400 kb breakpoint cluster region adjacent to the centromere on the short arm of chromosome 17. Importantly, our findings indicate that loss of p53 is not due to loss of chromosome 17, simple deletions of p53, or chromosome breakage near the p53 locus. The region between the centromere and HNPP on chromosome 17 is known to be rich in low copy repeats and homologous DNA sequences are dispersed throughout the genome. In somatic cells, these repeats can result in pairing of the same or different chromosomes; crossing over within these homologous regions can result in translocations or isochromosomes. Undoubtedly, this genetic mechanism plays an important role in the origin of structural anomalies of chromosome 17 in B-CLL.

Abstract The most common recognized cytogenetic change in B-CLL is a deletion involving band 13q14.3. Dewald found a heterozygous or homozygous 13q– in 92% of CLL patient samples using the FISH probe D13S319 that hybridizes to 13q14.3 (BJH 121:287, 2003). This contrasts with the usually normal conventional chromosome analysis (CCA) results partly because CLL cells divide infrequently but also because the 13q– is often visualized only by FISH (Stockero, Ca Genet Cytogenet 166:152, 2006). Mayr recently showed that CpG stimulation can reveal a chromosomally abnormal CLL clone in metaphase cells (Blood 107:742, 2006). The purpose of the present study was to compare the incidence of microdeletion and visible 13q deletion in CLL using interphase FISH and CCA after CpG culture on the same peripheral blood specimens. METHOD: We compared the chromosome 13 results by interphase FISH (fresh uncultured cells) and by a 20 metaphase CCA from 5–day CpG cultures. Our unselected CLL cohort (n=40) represented a typical and relatively high risk population: median age 61 (range 36–75), 48% CD38–positive, 62% ZAP–70 positive, and 48% IgVH unmutated. In addition 70% were previously treated for progressive disease, with all Rai stages represented. CpG RESULTS: By CCA of 40 patients, CpG stimulation revealed a clonal (multiple abnormal cells) or nonclonal (one abnormal cell) abnormal karyotype in 32 (80%) and a normal karyotype in 8 of the 40 patients (20%). Among the 32 abnormal cases, each chromosome 13 pair appeared normal in 17 and was abnormal in 15 (one was monosomy 13, 8 were 13q–, and 6 had a 13q translocation). CpG did not reveal a homozygous 13q abnormality in any patient. In total, CpG revealed a 13q abnormality in 15 of 40 (38%) patients (8 had multiple abnormal metaphases and 7 had only one abnormal metaphase). FISH RESULTS: By interphase FISH, 29 of 40 (73%) patients had a 13q–. The deletion was heterozygous in 18 patients, homozygous in 7, and mixed homo– and heterozygous in 4. Of the 18 with a heterozygous 13q loss by FISH, CpG revealed an abnormal 13 in only 8. Of the 11 patients with homozygous or mixed homo– and heterozygous 13q– by FISH, CpG revealed a heterozygous 13q abnormality in only 6. Of the 8 patients with a normal CpG karyotype, by FISH 5 had a 13q– in one or both 13s. Of the 9 patients with heterozygous 13q loss by CpG, the FISH result was 13q– in 4 and homozygous or mixed homo– and heterozygous 13q– in 5. Of the 6 with a chromosome 13 translocation by CpG, FISH revealed heterozygous 13q– in 4, mixed homo– and heterozygous 13q– in one, and in one case the 13q FISH result was within normal limits. In this latter case, CpG stimulation followed by metaphase FISH analysis confirmed that the apparently balanced translocation harbored a microdeletion 13q. Interphase FISH analysis confirmed the presence of a clonal deletion 13q in all 7 of the patients with a CpG metaphase result of a non-clonal (one metaphase cell) 13q abnormality. CONCLUSIONS: Even though CpG induces CLL metaphases in cell culture and reveals many chromosome abnormalities that cannot be identified by interphase FISH, cytogenetic evaluation of CLL by CpG alone significantly underestimates the incidence of 13q deletions. When a 20-cell analysis after CpG stimulation reveals a 13q abnromality in only one (apparently nonclonal) metaphase cell, concurrent interphase FISH analysis often confirms clonality for the 13q defect.

Abstract Background: Using fluorescent in-situ hybridization (FISH), a number of investigators have identified specific cytogenetic abnormalities that identify CLL patients with a more aggressive (17p-, 11q-) or indolent (13q-) disease course. Some have suggested patients who initially have a normal karyotype may acquire new chromosome abnormalities during the course of their disease. Since patients with specific cytogenetic abnormalities (17p-, 11q-) are less likely to respond to purine nucleoside analogues, such clonal evolution has potential implications for treatment as well as prognosis. No study has prospectively investigated the frequency of clonal evolution in a cohort of patients with newly diagnosed untreated CLL. Methods: Between 1994 and 2000, we enrolled 167 patients with previously untreated CLL seen at Mayo Clinic in a prospective trial evaluating the prognostic importance of cytogenetic abnormalities and clonal evolution detected by FISH. All patients provided a baseline blood specimen for FISH testing and follow-up specimens over the following 24 months. Other research samples from later timepoints were tested where available. Study participants were contacted by mail in 2004 to update vital and treatment status. Of 83 living responders, 70 (84%) indicated they would be willing to provide an additional follow-up sample for cytogenetic analysis of whom 48 have returned a sample to date. Results of clinical FISH testing during the follow-up interval were also abstracted. FISH was performed on interphase nuclei from blood as we have previously described (BJH 121:287). Results: Median age at diagnosis was 64. Median time from diagnosis to study enrollment was 3.3 months. 94% of patients had early stage disease at enrollment (88 Rai 0; 48 Rai I, 18 Rai II, 2 Rai III; 8 Rai IV). Median follow-up time from diagnosis for all 164 eligible study participants was 8.5 years (range: 0.33–22.9 yrs). As of last follow-up, 48% of patients have received treatment and 57 (35%) have died. 75% of patients had chromosome abnormalities on FISH testing at baseline. The frequency of individual cytogenetic abnormalities on baseline FISH analysis along with overall survival by hierarchical FISH risk category are shown in Table I. 106 patients had sequential samples for FISH analysis at least 2 years apart, 61 had samples at least 5 years apart, and 22 had samples at least 10 years apart. 15 patients had evidence of clonal evolution during follow up as evidenced by a new FISH anomaly not present on the baseline specimen. No clonal evolution was observed in the first 2 years of follow-up (n=106), however of 61 patients with samples at least 5 years apart, 14 (23%) had evidence of clonal evolution. Median time for development of a new cytogenetic abnormality among these patients was 9.3 years. Conclusions: Clonal evolution occurs during the course of disease for approximately 25% of patients with early stage CLL. Clonal evolution appears to occur at low frequency during the first 2 years of follow-up but increases in frequency after 5 years. This finding has potentially significant implications for prognosis and treatment of patients with CLL. FISH Risk Category* N (Baseline) Median Overall Survival (Years) * Difference between groups significant p=0.0038 13q- x 1 37 14.4 13q- x2 35 17 Normal Karyotype 40 13.2 12+ 24 11.1 11q- 12 8.6 17p- 10 10.5 6q- 2 4.1 Other 2 Not reached

Abstract Background: In 2006 Lenalidomide received FDA approval for myelodysplasia (MDS) with interstitial deletion 5q [del(5q)] based on conventional cytogenetic (CC) results before and after treatment. In clinical practice, FISH studies of interphase nuclei with probes for EGR1 are widely used to detect del(5q), but it is not known whether this method has similar efficacy as CC. Thus, we compared CC and FISH results for various forms of del(5q). Methods: We selected 145 patients (pts) in our cytogenetic database with del(5q). These pts had various forms of del(5q) and were representative of our clinical practice. This series included 9 pts with MDS that were studied before and after treatment with Lenalidomide. CC studies were done using standard procedures to analyze 20 metaphases from bone marrow. FISH studies were done on the same specimens using stored left-over cells and standard procedures for EGR1 (5q31.2) and D5S23/D5S721 (5p15.2). The cutoff for del(5q) for FISH was <6.0% based on 200 nuclei. Results: Among 145 pts, 104 were referred for MDS, 9 acute myeloid leukemia, 9 lymphoproliferative disorders, 7 myeloproliferative disorders and 16 without clinical information. A total of 116 pts had breakpoints at del(5)(q13q33), 17 del(5)(q15q33), 8 del(5)(q11.2~q31q31~q35), 3 del(5)(q22q33) and 1 del(5)(q13q22). Del(5q) was detected in 144 pts by both CC and FISH. One pt had del(5)(q13q22) in 20/20 metaphases but was normal by FISH. Percent abnormal cells by FISH was lower in 135/145 pts compared to CC (mean difference 22.5 ± 19.6, range −42 to 98). For 9 pts with MDS, 29 specimens were collected before and after treatment with Lenalidomide: 7 achieved complete cytogenetic remission (four relapsed) and 2 showed a decrease in percent abnormal metaphases. Results of CC and FISH for these 9 pts were concordant for detection of del(5q) and trend of tumor burden due to treatment for 28/29 specimens. The discordant specimen was collected 222 days after treatment; results of CC were normal but FISH was abnormal in 19% of nuclei. Among the 29 specimens, percent abnormal cells by FISH was lower than CC in 27 (mean difference 13.9% ± 22.5, range −19.0 to 73.5). Each of the 9 treated pts had only del(5q) prior to treatment, and was detected by both FISH and CC. After treatment, FISH did not detect a separate cytogenetic clone involving deletion 20q without del(5q) in 2 pts nor did it identify development of other anomalies in 3 pts. Conclusions: Results of CC and FISH were mostly concordant, but differed in 4 respects. First, 1/145 pts was normal by FISH but had del(5q) by CC. This pt had unusual breakpoints that did not result in loss of EGR1. Second, percentage of cells with del(5q) was usually lower by FISH than CC. This observation is likely related to a different mitotic rate of cells with del(5q) versus normal cells. Third, 1 specimen was abnormal by FISH but normal by CC after treatment, but 2 subsequent specimens were concordant. This discrepancy was likely due to interference of mitosis of del(5q) cells and residual non-proliferating disease. Fourth, 2 treated pts developed a separate cytogenetic clone without del(5q) and 3 developed other anomalies that could not be detected by FISH. We believe FISH for EGR1 is useful in clinical practice, but not a substitute for CC.

Purpose Retrospective studies suggest cytogenetic abnormalities detected by interphase fluorescent in situ hybridization (FISH) can identify patients with chronic lymphocytic leukemia (CLL) who will experience a more aggressive disease course. Other studies suggest that patients may acquire chromosome abnormalities during the course of their disease. There are minimal prospective data on the clinical utility of the widely used hierarchical FISH prognostic categories in patients with newly diagnosed early-stage CLL or the frequency of clonal evolution as determined by interphase FISH. Patients and Methods Between 1994 and 2002, we enrolled 159 patients with previously untreated CLL (83% Rai stage 0/I) on a prospective trial evaluating clonal evolution by FISH. Patients provided baseline and follow-up specimens for FISH testing during 2 to 12 years. Results Chromosomal abnormalities detected by FISH at study entry predicted overall survival. Eighteen patients experienced clonal evolution during follow-up. The rate of clonal evolution increased with duration of follow-up with only one occurrence in the first 2 years (n = 71; 1.4%) but 17 occurrences (n = 63; 27%) among patients tested after 5+ years. Clonal evolution occurred among 10% of ZAP-70–negative and 42% of ZAP-70–positive patients at 5+ years (P = .008). Conclusion This clinical trial confirms prospectively that cytogenetic abnormalities detected by FISH can predict overall survival for CLL patients at the time of diagnosis, but also suggests that many patients acquire new abnormalities during the course of their disease. Patients with higher ZAP-70 expression may be more likely to experience such clonal evolution. These findings have important implications for both clinical management and trials of early treatment for patients with high-risk, early-stage CLL.