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Abstract Background Making genomic data available at the point-of-care and for research is critical for the success of the Precision Medicine Initiative (PMI), a research initiative which seeks to change health care by “tak(ing) into account individual differences in people's genes, environments, and lifestyles.” The Office of the National Coordinator for Health Information Technology (ONC) led Sync for Genes, a program to develop standards that make genomic data available when and where it matters most. This article discusses lessons learned from recent Sync for Genes activities. Objectives The goals of Sync for Genes were to (1) demonstrate exchange of genomic data using health data standards, (2) provide feedback for refinement of health data standards, and (3) synthesize project experiences to support the integration of genomic data at the point-of-care and for research. Methods Four organizations participated in a program to test the Health Level Seven International (HL7®) Fast Healthcare Interoperability Resources (FHIR®) standard, which supports sharing genomic data. ONC provided access to subject matter experts, resources, tools, and technical guidance to support testing activities. Three of the four organizations participated in HL7 FHIR Connectathons to test FHIR's ability to exchange genomic diagnostic reports. Results The organizations successfully demonstrated exchange of genomic diagnostic reports using FHIR. The feedback and artifacts that resulted from these activities were shared with HL7 and made publicly available. Four areas were identified as important considerations for similar projects: (1) FHIR proficiency, (2) developer support, (3) project scope, and (4) bridging health information technology and genomic expertise. Conclusion Precision medicine is a rapidly evolving field, and there is opportunity to continue maturing health data standards for the exchange of necessary genomic data, increasing the likelihood that the standard supports the needs of users.

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. Several studies have recently reported on the occurrence of a JAK2 V617F mutation in myeloid cells from the majority of patients with polycythemia vera (PV). The clinical relevance of this novel observation is currently under study. Similarly, there is limited information regarding the correlation of JAK2 V617F mutational status and neutrophil PRV-1 transcript level in PV, secondary polycythemia (SP) and other myeloproliferative disorders (MPD). METHODS. In a single institutional study, mutation screening for JAK2 V617F was performed in DNA derived from archived blood granulocytes from 63 consecutive patients with PV in whom current diagnostic criteria were strictly applied and diagnosis confirmed by bone marrow histology. Concomitant quantitative analysis of neutrophil PRV-1 expression was performed in 28 of the 63 patients and the results of both JAK2 V617F and PRV-1 analysis in these 28 patients were compared with those from another 22 patients with essential thrombocythemia (ET), 10 with myelofibrosis with myeloid metaplasia (MMM), 19 with SP, and 11 healthy volunteers. RESULTS. The 63 patients with PV were followed for a median of 33 months (range 3–324) from the time of initial diagnosis. During this period, 3 patients have died including 2 from acute myeloid leukemia (AML). Disease transformation into either AML or MMM has occurred in 2 and 4 patients, respectively. The median time from diagnosis to the time of mutation analysis was 12 months (range 0–306). JAK2 V617F was detected in 58 of the 63 patients with PV (92%; homozygous in 21%). The clinical phenotype of the 5 patients with wild-type allele was otherwise typical for the disease. A statistical comparison between JAK2 V617F heterozygotes (n=45) and homozygotes (n=13) in PV did not reveal significant associations in regards to age, gender, leukocyte or platelet count at diagnosis, disease duration, or the incidences of thrombosis or bleeding. However, JAK2 V617F homozygotes, compared to their heterozygote counterparts, displayed significantly higher hemoglobin level at diagnosis (p=0.001), increased incidence of pruritus (69% vs. 38%; p=0.04), and a higher rate of fibrotic transformation (23% vs. 2%; p=0.009). The overall (homozygous) JAK2 V617F mutational frequencies in the other study patients with ET, MMM, SP, and healthy controls were 55% (0%), 30% (0%), 0%, and 0%, respectively. The corresponding figures for increased PRV-1 expression in these patients were 18%, 20%, 21%, and 9% as compared to 89% in PV. In patients with either ET or MMM, the likelihood of detecting JAK2 V617F was significantly higher in the presence of an increased PRV-1 expression (83% vs. 38%; p=0.05). Similarly, in patients with PV, homozygous as compared to heterozygous JAK2 V617F correlated with higher levels of PRV-1 expression (p=0.07). CONCLUSIONS. The current clinical study demonstrates a mutant allele dose effect on erythrocytosis, PV-characteristic clinical features, and granulocyte PRV-1 expression in PV. Furthermore, compared to the PRV-1 assay, mutation screening for JAK2 V617F displayed greater specificity in distinguishing PV from SP.

Abstract Background: In essential thrombocythemia (ET), homozygosity for the JAK2 V617F mutant allele is rare despite the frequent (23–57%) occurrence of the heterozygous state. This is in direct contrast with the situation in polycythemia vera (PV) where homozygosity for the mutation is seen in 25–33% of the cases. The particular observation is consistent with what has been observed in vitro as well as in animal studies where JAK2 V617F has been associated with induction of erythropoietin hypersensitivity in cell lines and erythrocytosis in mice (James et al. Nature2005;434:1144). Therefore, regardless of whether or not JAK2 V617F represents a primary event in myeloproliferative disorders, it is probable that it promotes a PV phenotype. In the current study, we explore this hypothesis as well as investigate the long-term prognostic relevance of the mutation in a well defined cohort of patients with ET seen at a single institution. Methods: The diagnosis of ET was according to the WHO classification of tumors (Jaffe ES et al. Lyon, IARC press, 2001). All patients were seen at the Mayo Clinic and patient characteristics and subsequent clinical events were determined both retrospectively (chart review) and prospectively (by means of a questionnaire sent both to patients and their primary doctors). In addition date and cause of death were confirmed by probing both the social security index and the national death index. DNA for mutation screening was derived and sequenced from either archived unsorted cells from the bone marrow or prospectively collected peripheral blood granulocytes according to previously published methods (Levine et al. Cancer Cell, 2005;7:387). Results: The study cohort consisted of 150 consecutive patients with ET including 99 females and 51 males (median age 49.8 years; range 16.8–98). Overall, JAK2 V617F was detected in 73 patients (48.7%) but none were homozygous for the mutant allele. At diagnosis, ET patients with JAK2 V617F were significantly older (median age of 53.5 vs. 42.3; p=0.02) and also displayed higher readings of both hemoglobin level (p=0.0002) and leukocyte count (p=0.005). All study patients have now been followed for a median of 11.4 years from diagnosis (range 0–32.7) and follow-up period was similar between the two mutational categories (p=0.53). During this period, 32 patients have died and the median survival for the entire group was 19.3 years; 21.9 and 17.7 years in the absence and presence of the mutation, respectively (p=0.09). Disease transformation into acute leukemia, myelofibrosis, and PV occurred in 6 (4%), 15 (10%), and 8 (5.3%) patients, respectively, and the only significant difference between the two mutational categories in this regard was the higher incidence of transformation into PV in patients with the mutation (p=0.02). Multivariate analysis identified age, hemoglobin level, and thrombosis history at diagnosis but not JAK2 V617F mutational status as independent risk factors for survival. Conclusion: Although the presence of JAK2 V617F in ET appears to promote a PV phenotype, it might not carry treatment-relevant information.

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 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.

Abstract Certain normal and malignant B-cells rely upon “tonic” B-cell receptor (BCR)-mediated survival signals. BCR signaling induces the phosphorylation of the associated Ig alpha and beta proteins and the recruitment and activation of spleen tyrosine kinase (SYK); thereafter, SYK initiates downstream events and amplifies the original BCR signal. A subset of DLBCL exhibits coordinate overexpression of BCR pathway components, including SYK, and relies upon tonic BCR signaling. FosD is an orally available inhibitor of SYK under development for rheumatoid arthritis, and has demonstrated significant in vitro activity against BCR-dependent NHLs. We have performed the first clinical trial of the oral SYK inhibitor, FosD, in patients (pts) with a variety of relapsed or refractory B-cell non-Hodgkin’s lymphomas. We first enrolled 13 pts in a Phase I component, exploring two dose levels (200 and 250 mg BID) of FosD. Pts (Follicular [FL], 5; CLL/SLL, 2; mantle [MCL], 3; DLBCL, 3) were heavily pretreated. On the first day of FosD dosing at 200 mg and 250 mg, the maximum plasma concentrations were 668 ± 258 ng/mL and 1020 ± 781 ng/mL, and the AUC0–4 estimates were 1800 ± 602 ng*h/mL and 2590 ± 1900 ng*h/mL, respectively. Plasma concentrations increased approximately 2-fold with continued administration, but beyond Day 29 there was no apparent change in FosD concentrations measured over time. Dose limiting toxicity in Phase I was neutropenia; and 200 mg BID was chosen for Phase II evaluation. We then enrolled 68 pts with relapsed/refractory NHL in 3 separate disease cohorts: DLBCL (23); FL (21) and other B-cell NHL (24) including SLL/CLL (11), MCL (9) marginal zone/MALT (3) and lymphoplasmacytic NHL (1). The median age of pts in phase II was 61 (range 41–87); pts received a median of 5 prior therapies, including ASCT (16; 12 with DLBCL) and radioimmunotherapy (8). FosD was overall very well tolerated; there were 4 cases of febrile neutropenia reported. Eight pts required dose modification for neutropenia (2), hypertension (2), liver function test abnormalities (2), fever (1) and mucositis (1). Six pts withdrew before initial response evaluation (AE, 5; noncompliance, 1). Best responses by disease were: DLBCL 21% (4PR; 1CR); SLL/CLL 54% (6PR); FL 10% (2PR); MCL 11% (1PR). Stable disease was observed in an additional 23 patients, including 12 with FL, 4 with DLBCL, 4 with MCL, 2 with CLL/SLL and 1 with MALT. As of 7/15/08, 16 pts were treated for more than 200 days, and 14 pts remain on study. Median PFS is 4.5 months, and only 5 pts have died (4 with DLBCL of disease progression and 1 with CLL of infection following therapy). Several pts with CLL/SLL experienced a transient increase in circulating lymphocytes in the setting of responding nodal disease. Of the 6 responding pts with CLL, Zap-70 status was positive (3), negative (1) and unknown (2); cytogenetics were trisomy 12 (1), del13 (1), normal (1), complex (1) and unknown (2). We conclude that disrupting BCR-induced signaling by inhibiting SYK kinase represents a safe and well-tolerated novel therapeutic approach to NHL. In addition to significant responses in DLBCL and CLL/SLL, prolonged stable disease was observed in pts with FL. FosD should be developed further, as a single agent and in rational combinations, for BCR-dependent B-cell NHLs.

Abstract Background: This is an initial report of a correlative laboratory study of an ECOG clinical trial, E2903, to investigate an array of prognostic factors that are potentially associated with the clinical outcome in previously treated, high risk Chronic Lymphocytic Leukemia (CLL) patients. The ultimate goal of this study is to develop a prognostic model for this group of patients. Method: We have previously reported (ASH, 2007) on initial clinical and toxicity results using a combination of PCR followed by Campath. To date we have clinical response data on 37 patients and have conducted correlative prognostic factor analysis on these patients at entry to the study. The prognostic parameters assessed were fluorescence interphase hybridization (FISH) panel, CD38, ZAP-70 and IGVH (mutated vs. unmutated) status and plasma levels of pro- and anti-angiogenic cytokines. In addition, features such as clinical stage, age, sex and laboratory parameters including total white blood count, absolute lymphocyte count (ALC) and beta 2 microglobulin (b2m) were studied as predictors of response to PCR. Results: As of July 2008, the response data for the first 41 (the accrual goal is 110) patients who started the induction treatment are available. Of these 41, 3 patients are ineligible and one patient had no response data. The median age was 63 (range 51–76) with 29 males and 8 females, median b2m was 4 (range 2–28), 11 (31%) were Rai stage 0–2, and 26 (69%) were Rai 3–4. The median number of prior treatments was 2 (range 1–5); 15 of 23 (65%) patients tested for IGVH were unmutated, while on FISH analysis 4 had del 6q, 8 had del 11q, 4 had del 17p and 30 patients had complex FISH defects (>1 defect). Despite the prevalence of high risk features in this cohort of patients, 17 out of 37 responded (best response=partial response), 18 were stable and 2 had progressive disease with an overall response rate of 46%. Prognostic parameters most strongly associated with a clinical response included CD38 negative (p=0.008), ZAP-70 negative status (p=0.01) and mutated status (p=0.03). Responses were independent of age, sex, b2m, ALC, number of prior therapy or FISH defects. Responders showed a prolonged progression-free survival (PFS) compared to the PFS of non-responders (i.e., 2-yr PFS 29% vs. 0%, respectively, p<0.0001). Prognostic parameters most strongly associated with PFS included being both ZAP-70 negative (p=0.0009) and CD38 negative (p=0.004). Also, ZAP-70 negative and CD38 negative status was a strong predictor of favorable PFS (p=0.002). Mutated status was borderline significant (p=0.07) in univariate analysis and there was no obvious correlation of baseline angiogenic cytokine plasma levels with PFS. These results also hold in a multivariable Cox model. In the Cox model, mutated status was significantly associated with PFS (HR=0.2, p=0.01). Conclusion: We believe that the PCR regimen is highly effective at inducing responses in previously treated relapsed or refractory CLL patients who carry high risk prognostic markers. Thus, while no CRs were seen all but 2 patients had a PR or were stable after 6 cycles of PCR. The prognostic parameters most strongly associated with predictors of clinical outcome appear to be ZAP-70, CD38 and mutation status. The ability to attain a response also appears to be important for achieving a prolonged PFS. In summary, we believe that the strong clinical activity of PCR in a high risk group of CLL patients provides more evidence for pursuing the regimen as a platform for other combinations. In addition, we have generated a first level prognostic model that will continue to be refined as we proceed to complete this trial.

Abstract BACKGROUND: Karyotype analysis of metaphase cells for discovering genome-wide cytogenetic abnormalities has played an important role in prognosis and counseling of patients with chronic lymphocytic leukemia (CLL). Due to the low proliferative rate of CLL B cells, karyotype analysis has been of limited clinical utility in this disease. Thus, fluorescence in situ hybridization (FISH) on interphase cells has replaced karyotype analysis for the assessment of recurrent genetic changes in CLL. Recently, immunostimulatory CpG-oligodeoxynucleotides (CpG) have been shown to induce primary CLL B cell proliferation via engagement of toll-like receptor 9 (TLR9), thereby rendering karyotype analysis feasible and providing for a useful and reliable method to discover new cytogenetic abnormalities in CLL B cells. However, we and others have shown that CpG stimulation of CLL and normal B cells also induces robust expression of activation induced cytidine deaminase (AID), a mutagenic enzyme that is linked to the development of cytogenetic abnormalities. It is therefore plausible that cytogenetic abnormalities observed following CpG stimulation of CLL B cells may not reflect de novo abnormalities but rather reflect CpG induced-AID mediated cytogenetic alterations. We began to address this possibility by assessing the effects of CpG stimulation on the karyotypes of normal blood B cells. HYPOTHESIS: Since CpG stimulation induces AID expression in CLL and normal B cells, we hypothesize that: robust B cell activation by CpG could result in cytogenetic abnormalities thereby contributing to lymphomagenesis; and if CpG induces cytogenetic abnormalities that are linked to disease initiation and/or progression, such information would be of great value in the interpretation of cytogenetic data obtained from CpG-stimulated CLL B cells. METHODS: We performed interphase FISH analysis on freshly isolated blood B cells, and metaphase karyotype analysis on CpG stimulated B cells from 17 healthy blood donors. Pure (>98%) B cells obtained by negative selection, were hybridized at isolation with a probe set to identify deletion 6q, 11q, 13q, and 17p, trisomy 12, and IGH gene rearrangements, and cells were also stimulated for 5 days with 10 μg/ml CpG (CpG 2006, synthesized in-house) plus IL-2 and IL-15 for metaphase analysis. At least 20 post-CpG metaphase spreads were analyzed for each sample and abnormalities were considered clonal using the accepted convention (gains or translocations are present in two or more cells and loss present in at least 3 cells). The expression of AID in the B cells pre- and post-CpG stimulation was assessed by RT-PCR. RESULTS: FISH analysis of freshly isolated B cells showed that all 17 subjects were normal. After CpG stimulation, all samples examined exhibited strong AID expression as revealed by RT-PCR. By karyotype analysis, none of the 17 CpG stimulated samples showed evidence of clonal abnormalities. However, one or more nonclonal chromosomal abnormalities were observed in 7 out of 17 (41.2%) samples. Among those 7 samples, 4 samples had 1 abnormal metaphase out of a minimum of 20 examined while the other 3 samples exhibited two or more cells, each with distinct abnormalities. Some of those nonclonal abnormalities have also been observed in CLL (i.e., trisomy 3, trisomy 12 and 14q32 rearrangements). This is a much higher incidence of nonclonal abnormalities than has been seen with peripheral blood cells cultured by other methods. CONCLUSION: Our data have significance for the biology of CLL and for clinical practice. First, our data suggest that signaling through TLR9, and perhaps other receptors that likewise induce robust B cell AID expression, may represent a natural mechanism by which B lineage cells acquire chromosomal abnormalities during normal immune responses that may predispose toward neoplastic transformation. Second, given that none of the CpG-induced metaphases in normal B cells resulted in clonal abnormalities in the 5 day timeframe, our data also validate the clinical utility of CpG as a valuable tool for the cytogenetic analysis of CLL B cells since only clonal abnormalities are currently scored. However, it remains possible that non-clonal abnormalities seen in CpG-stimulated CLL B cells may be relevant to the disease process if future work shows that CpG stimulation uncovers recurring non-clonal defects of chromosomes/genes associated with tumorigenic events.