Smoley, Stephanie A.

AbstractThis case report underlines the importance of molecular characterization of genomic duplications and other structural variants in the prenatal setting to guide clinical interpretation, genetic counseling, and perinatal medical care.

Purpose: Acute myeloid leukemia (AML) is the most common acute leukemia in adults, affecting approximately 20,000 patients annually in the United States. AML genetic subtypes, as defined by the World Health Organization (WHO), are identified through fluorescence in situ hybridization (FISH), conventional chromosome analysis, and sequencing techniques. Mate pair sequencing (MPseq) is a next generation sequencing (NGS) technology optimized to detect genome wide structural variants and copy number alterations at high resolution. Our study goal was to investigate the prognostic value of MPseq in comparison to FISH, chromosome, and sequencing studies in the evaluation of AML patients. Methods: We performed a prospective study using blood and bone marrow samples from 105 patients with a diagnosis of AML, using MPseq, along with chromosome, FISH, and NGS or PCR studies to detect small mutations. Cytogenetic and molecular genetic results were correlated with MPseq findings. We also analyzed the MPseq data for chromoplexy, chromothripsis, and progressive complexity. Junction and copy number burden, the incidence of structural variation in the genome and the percent of the genome with aberrant copy number, were evaluated. Overall survival statistics were stratified by AML subtypes and observed anomalies. Results: Although structural variants in AML were characterized at a high resolution using MPseq when compared to conventional cytogenetic methods, risk stratification using current European Leukemia Net (ELN) guidelines was not improved by MPseq. The cohorts involving 5q and/or 7q deletions exhibited high levels of genomic complexity when compared to normal karyotype AML (NK-AML). The incidence of copy number gains, losses and junctions was greatest in 5q and 7q co-deletions (5q/7q) (16.5, 25.0, 69.3) and 5q deletions (5q) (9.8, 16.7, 31.6) subtypes compared to 7q deletions (3.4, 7.0, 6.7) and NK-AML (2.6, 4.3, 3.8) (p<0.001) subtypes. Chromoplexy, chromothripsis, and progressive structural complexity were detected in most samples with 5q deletions and 5q/7q co-deletions, but absent in samples with 7q or NK-AML. Biallelic inactivation of TP53 by sequencing mutation and/or deletion was common in the 5q/7q co-deletion (14/18 cases) and 5q deletion cohorts (7/10 cases), rare in the 7q deletion cohort (1/11 cases), and absent in the NK-AML (n=44) cohort. The median OS was significantly worse for patients with 5q/7q deletions (122.5 days) and 5q deletions (248 days) compared to NK-AML (413.5 days; p<0.001 and p=0.017, respectively) and between 5q/7q deletions and 7q deletions (370.5 days; p<0.001). No significant difference was observed between 5q/7q and 5q deletion subtypes, between NK and 7q deletion subtypes and between 5q and 7q deletion subtypes. The median OS was also significantly shorter for patients with TP53 alterations compared to patients with normal TP53 status. Patients with chromoplexy, chromothripsis and/or progressive structural complexity identified by MPseq had a significantly shorter median OS compared to patients without these features. (p<0.0001) Discussion: Risk stratifications based on current guidelines using cytogenetic and sequencing results were not adjusted due to MPseq results, which is not surprising when primary abnormalities are observed by conventional cytogenetic methods. NK-AML cases did not appear to benefit from a high resolution genomic evaluation. However, MPseq added value when structural variation required additional characterization, detecting novel rearrangements, such as a KAT6A/SORBS3 fusion. Lastly, we recognized common mischaracterizations made by conventional chromosome studies - including missed TP53 deletions in 7 cases, 5q/7q deletions misinterpreted as monosomies, cryptic NUP98 rearrangements, and unappreciated genomic complexity correlating with poor OS. These mischaracterizations challenge the use of conventional chromosome studies as a gold standard without accompanying FISH or MPseq studies. MPseq, similar to other structural methodologies such as optical mapping and long read sequencing, should be considered important complements to standard cytologic techniques given the important additional genomic information obtained. The additional structural variant characterization will be critical in paving the way for genomic discovery with the overall goal of improving prognostication for patient care. Figure Disclosures Vasmatzis: WholeGenome LLC.: Other: Owner; Mayo Clinic: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Background: Cytogenetic abnormalities detected by fluorescence in situ hybridization (FISH) are known to differentially impact prognosis in patients with chronic lymphocytic leukemia (CLL). Deletion6q23 (del6q23) has been reported in ~2-3% of CLL patients at diagnosis; however, its prognostic significance remains indeterminate. Methods: We identified previously untreated CLL patients from the Mayo Clinic CLL Database seen between 1/1995 - 2/2020 who had FISH performed. The clinical characteristics, concomitant cytogenetic abnormalities, time to first therapy (TTFT), and overall survival (OS) were compared between patients with del6q23 to those without this abnormality. TTFT was analyzed from FISH date until treatment or last known untreated date, accounting for competing risk of death. OS was analyzed using Kaplan-Meier methods from FISH date. In a matched (CLL-IPI risk score and sex) analysis, Cox regression analysis was used to determine the association between TTFT/OS and presence of del6q23. The Mayo Clinic IRB approved this study. Results: A total of 3101 CLL patients were identified; their median age at diagnosis was 64 years and 66% were male. Of these patients, 42 (1.3%) had evidence of del6q23 by FISH. Compared to patients without del6q23, patients with del6q23 carried a more adverse clinical and cytogenetic profile at the time of diagnosis including advanced Rai stage (22% Rai III/IV vs. 9% Rai III/IV; P = 0.002), unmutated IGHV genes (91% vs. 43%; P < 0.001), and high/very high risk CLL-IPI (50% vs. 27%; P < 0.001). Del6q23 was the sole abnormality in 13 patients (31%). Of the remaining patients, del6q23 was concomitantly present with del13q in 8 patients (19%), and with either del11q or del17p in 21 patients (50%). Of the 42 patients with del6q23, 29 received first line therapy: chemoimmunotherapy in 16 patients, BTK inhibitor in 7 patients, and an anti-CD20 monoclonal antibody alone in 6 patients. The median follow-up of the entire cohort was 7.3 years. The median TTFT in patients with del6q23 was 0.7 years while the 5-year OS was 76%. Patients with del6q23 demonstrated significantly shorter TTFT and reduced OS when compared to patients without del6q23 (Figures 1A and 1B). The shorter TTFT was also evident for patients with del6q23 compared to patients without del6q23 divided according to the Dohner risk category (Figure 2A), although there was no difference in OS between these groups of patients (Figure 2B). Because del6q23 patients were enriched for other unfavorable prognostic markers, we identified four control patients without del6q23, matched by CLL-IPI risk score and sex, to each treatment-naïve patient with del6q23. Patients with del6q23 showed a borderline statistically significant shorter TTFT (median 0.7 years versus 3.9 years; P = 0.07) and a nonsignificant association with OS (median 10.9 years versus 10.3 years; P = 0.28). Conclusion: Del6q23 by FISH in previously untreated CLL represents an uncommon abnormality. When compared to patients without del6q23, patients with del6q23 tend to have more adverse clinical and cytogenetic profiles at baseline, which likely confer a worse prognosis as reflected by shorter TTFT compared to patients without this abnormality. Figure Disclosures Ding: Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; DTRM: Research Funding; Beigene: Membership on an entity's Board of Directors or advisory committees; alexion: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Membership on an entity's Board of Directors or advisory committees; Octapharma: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding; Astra Zeneca: Research Funding. Wang:Novartis: Research Funding; Innocare: Research Funding; Incyte: Research Funding. Braggio:DASA: Consultancy; Bayer: Other: Stock Owner; Acerta Pharma: Research Funding. Kay:Sunesis: Research Funding; Abbvie: Research Funding; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno Theraputics: Membership on an entity's Board of Directors or advisory committees; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Rigel: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Cytomx: Membership on an entity's Board of Directors or advisory committees; Agios Pharma: Membership on an entity's Board of Directors or advisory committees; Acerta Pharma: Research Funding; MEI Pharma: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees. Kenderian:Juno: Research Funding; Gilead: Research Funding; Lentigen: Research Funding; MorphoSys: Research Funding; Sunesis: Research Funding; Kite: Research Funding; Novartis: Patents & Royalties, Research Funding; Torque: Consultancy; Humanigen: Consultancy, Patents & Royalties, Research Funding; Mettaforge: Patents & Royalties; Tolero: Research Funding; BMS: Research Funding. Parikh:Ascentage Pharma: Research Funding; Merck: Research Funding; AbbVie: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; TG Therapeutics: Research Funding; Pharmacyclics: Honoraria, Research Funding; GlaxoSmithKline: Honoraria; Janssen: Honoraria, Research Funding; MorphoSys: Research Funding; Genentech: Honoraria; Verastem Oncology: Honoraria.

A fetus harboring a duplication of SMAD2 exons 1-6 presented with dextrocardia and pulmonary hypoplasia. Mate-pair sequencing revealed the duplication to be in inverted tandem orientation to the wild-type SMAD2 allele, disrupting its sequence and decreasing expression. These observations suggest SMAD2 to be responsible for the fetal dextrocardia.

e22002 Background: Mesenchymal stem cells (MSC) residing in the marrow support hematopoiesis and protect cancer cells from undergoing cell death induced by chemotherapy. Recent reports have described clonal cytogenetic abnormalities in the MSC of acute myeloid leukemia and myelodysplastic syndrome patients. To determine if cytogenetic abnormalities are present in MSC from CLL patients, we analyzed karyotypes of MSC from 13 CLL patients and 5 normal subjects. Methods: Stromal cells from marrow core biopsies of 13 CLL patients and 5 normal control subjects were isolated, cultured and confirmed as MSC based on their immunophenotype and capacity to differentiate into three lineages. After 3–4 non-stimulated cell culture passages, the karyotype was analyzed in 5–40 metaphase cells from each subject Abnormalities were considered clonal using the accepted convention of the same chromosomal gain or rearrangement in 2 or more cells or loss in at least 3 cells. For each CLL patient, interphase FISH analysis to detect the common CLL-associated abnormalities was performed on freshly isolated peripheral blood mononuclear cells (PBMC). Results: Clonal cytogenetic abnormalities of the cultured MSC were observed in 6 of 13 CLL patients (46%) and 3 of 5 control subjects (60%). The 6 CLL MSC had different clonal abnormalities than the PBMC CLL FISH studies. One CLL MSC exhibited trisomy 5, one exhibited trisomy 8, and one had monosomy X clone. One MSC had a balanced t(1;16). One MSC had a balanced t(3;12) and a del(3p), and one had a complex karyotype with 4 unbalanced rearrangements. The clonal aberrations observed in the 3 controls included a del(3p) in two cases, and in one case an inv(5) and a del(10q). There was no correlation of the chromosomal abnormalities in CLL MSC with clinical stage. Conclusions: Marrow MSC derived from CLL patients and normal subjects do show an array of cytogenetic abnormalities including clonal chromosomal abnormalities. However the genetic abnormalities found in both CLL and normal MSC could represent acquired genomic instability associated with advanced age, rather than oncogenesis associated with CLL. [Table: see text]

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 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 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.