Dyke, Daniel Van

Common sex chromosome aneuploidies (SCAs) often present with cognitive and cardiovascular dysfunction in humans. To address SCA effects on gene expression and DNA methylation in relevant cell types, we differentiated neural precursor cells (NPCs) and cardiomyocytes (CMs) from human induced pluripotent stem cells (hiPSCs) with different numbers of sex chromosomes, including isogenic and independent lines. As expected, the expression of genes that escape X inactivation (escapees) mostly increases with the number of inactive X chromosomes (Xi). However, allelic analysis shows dampening of escapees specifically on the Xi in XXY compared to XX in both NPCs and CMs, revealing a novel type of dosage compensation in SCA. In contrast, Y-linked gene expression is higher in XXY versus XY NPCs, but the opposite is observed in CMs. This may explain the greater number of differentially expressed autosomal genes in NPCs versus CMs with an added Y chromosome, while effects of added X chromosomes are similar between cell types. Concordantly, changes in autosomal DNA methylation are mainly driven by the presence of a Y chromosome. These findings highlight the cell-type specificity of sex-linked and autosomal gene regulation in SCA conditions.

Abstract The utility of the chronic lymphocytic leukemia-international prognostic index (CLL-IPI) in predicting outcomes of individuals with Rai 0 stage CLL and monoclonal B-cell lymphocytosis (MBL) is unclear. We identified 969 individuals (415 MBL and 554 Rai 0 CLL; median age, 64 years; 65% men) seen at Mayo Clinic between 1 January 2001 and 1 October 2018, and ascertained time to first therapy (TTFT) and overall survival (OS). After a median follow up of 7 years, the risk of disease progression needing therapy was 2.9%/y for MBL (median, not reached) and 5%/y for Rai 0 CLL (median, 10.4 years). Among patients with low, intermediate, and high/very high-risk CLL-IPI risk groups, the estimated 5-year risk of TTFT was 13.5%, 30%, and 58%, respectively, P< .0001 (c-statistic = 0.69); and the estimated 5-year OS was 96.3%, 91.5%, and 76%, respectively, P< .0001 (c-statistic = 0.65). In a multivariable analysis of absolute B-cell count with individual factors of the CLL-IPI, the absolute B-cell count was associated with shorter TTFT (hazard ratio [HR] for each 10 × 109/L increase: 1.31; P< .0001) and shorter OS (HR: 1.1; P = .02). The OS of the entire cohort was similar to that of the age- and sex-matched general population of Minnesota (P = .17), although Rai 0 CLL patients with high and very high-risk CLL-IPI score had significantly shorter OS (P= .01 and P= .0001, respectively). The results of this study demonstrate the ability of CLL-IPI to predict time from diagnosis to first treatment (an end point not affected by therapy) in a large cohort of patients whose only manifestation of disease is a circulating clonal lymphocyte population.

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.

7055 Introduction: Cytogenetic abnormalities have been shown to be the most important determinant of prognosis in patients with acute myeloid leukemia (AML). In AML, trisomy 13 has been placed in the intermediate prognostic category although recent reports associate it with a more dismal outcome. The number of cases reported in the literature are relatively few, we reviewed our experience with trisomy 13 in hematologic malignancies to determine its significance and prognosis. Methods: We conducted a clinicopathological review of cases seen in our institution from January of 1990 to October of 2006. Our cytogenetic records were searched for patients, who had a hematological disorder whose non-stimulated bone marrow karyotype was found to have trisomy 13 as a sole abnormality. In men the absence of the Y chromosome was not regarded as a separate abnormality. Results: A total of 27 patients were identified. The median age was 76 years (range 25–87), 22/27 were male. The number of cases and diseases identified were 15 with AML, 5 with a myelodysplastic syndrome, 4 with an uncategorized myelodysplastic/myeloproliferative disorder, 2 with acute lymphoblastic leukemia and 1 with a chronic myeloproliferative disorder. Of the AML group 7 were M0 according to the FAB classification, 2 patients were identified to have M4 and another two M2. A single case each of M1 and M6 was also identified. In these 15 patients, 9 underwent induction chemotherapy, which consisted of intermediate dose Ara-C in 3 cases, anthracycline plus Ara-C in 5 patients, and daunorubicin, vincristine and prednisone in 1. Another 3 patients were given palliative treatment. For the rest of the subjects the type of treatment was unknown. A CR was obtained in 6 patients, 1 patient underwent a myeloablative stem cell transplant. Death has occurred in 24 patients, the median survival of the entire group was 5.4 months. In the patients with AML the median survival was 4.7 months, of the patients who achieved a CR the median survival was 9.5 months. Conclusions: We describe one of the largest series reported to date of trisomy 13 in hematologic malignancies and found that trisomy 13 is associated with male gender and an older age at presentation, as well as a poor prognosis. In AML its presence is associated with an FAB-M0 phenotype and its occurrence should be regarded as a high-risk feature. No significant financial relationships to disclose.

Abstract Purine nucleoside combination therapy, with and without immunotherapy has become the standard treatment approach to CLL. Past approaches, other than stem cell transplantation, have not been successful in curing the disease. Inherent resistance of CLL precursors, impaired immune surveillance (T-cells, NK cells) and microenvironmental abnormalities may all play a role in the ability to effectively eradicate leukemia. PCR has been successfully employed for previously treated (Lamanna 2006) and previously untreated (Kay 2007) patients with CLL. In addition, CA has been added to FCR (Wierda 2006) to treat relapsed/refractory CLL disease. We designed a study to evaluate PCR followed by CA for relapsed/refractory CLL. This ECOG study includes, in addition to response criteria, molecular correlates (FISH anomalies and CpG karyotype), risk parameters analysis, including CD38, ZAP-70 and IgVH mutational status. We assessed the baseline and sequential changes in the angiogenic status of patients by measuring plasma VEGF, bFGF, thrombospondin and their immune status via T-cell repertoire. From September 2004 to September 2006, 32 patients with relapsed/refractory CLL were enrolled in the protocol. The protocol combined 2 regimens (PCR and CA). Step 1, PCR, was planned for 6 monthly cycles. Steps 2 and 3 consisted of short term CA (4 weeks) or standard CA (18 weeks) depending upon Step 1 response. There were 3 ineligible, 1 not evaluated, and 28 evaluable patients. FISH anomalies revealed that 8 patients had a homozygous or heterozygous 13q-defect, 9 had complex FISH defects (more than one FISH anomaly), 5 patients had FISH anomalies that included del(17p13.1), 4 patients had FISH anomalies that included 6q-and 3 had FISH anomalies that included 11q-. CD38 was positive in 10 patients, 11 were ZAP-70 positive and 12 of 19 patients tested had unmutated type clones. The planned 6 cycles of Step 1 (PCR) was administered to 14/32 patients (44%), the others receiving 0–5 cycles. All responses were considered partial responses (11/29; 37.9%), with 2 patients (6.9%) demonstrating progressive disease. However, patients receiving the planned 6 cycles of PCR, achieved a partial response of 71% (10/14) with 2 cases of tumor lysis syndrome. Toxicity (Step 1-PCR) was predominately myelosuppressive (Grade 1–4). A case of chronic idiopathic polyneuropathy and a case of “paraneoplastic pemphigus” were reported. There were 4 deaths. CMV reactivation was diagnosed in 2 patients, neither of which was fatal. During Step 2 (CA), short-term administration in one patient led to grade 3 skin toxicity, which limited administration. During long-term administration of CA (PR, PD, SD) (n=7), infectious complications occurred in 6 patients all of whom developed grade 4 lymphopenia. The median survival for the entire group was 15 months. In conclusion, a significant number of heavily pre-treated and poor prognosis patients with CLL will respond to PCR. Further analysis will determine whether a significant number of patients will achieve a higher level of response and longer survival after CA administration. Studies of association of outcome with the risk parameters are ongoing and will be presented with the clinical response data.

Abstract Abstract 1246 Poster Board I-268 B-cell chronic lymphocytic leukemia (B-CLL) is characterized by a variable clinical course. Genomic aberrations as detected by FISH analysis and the mutational status of the IgVH genes have been found to have prognostic clinical significance with unmutated IgVH genes associated with clinically more aggressive disease. Previous studies have suggested that unmutated IgVH B-CLL is associated with increased chromosomal instability and increased chromosomal aberrations leading to accelerated disease progression. We recently performed a high-throughput, high-resolution array-based comparative genomic hybridization (aCGH) in a cohort of B-CLL patients. Fifty-eight patients have been analyzed by using the Sureprint G3 microarray (one million probes, Agilent). Our working hypothesis was that we would see increased genomic aberrations and instability in the unmutated IgVH group. The total number of copy number aberrations and total megabases (Mb) deleted or gained per genome were used as a surrogate marker for genomic instability. Variations in chromosomal aberrations in various VH families were also evaluated. B-CLL with unmutated IgVH genes utilized members of the VH1, VH3, VH4 and VH5 families with VH1-69 the most prevalent. B-CLL with mutated IgVH genes utilized VH families VH1, VH2, VH3, VH4, and VH6. Considerable variation was seen in the number of genomic abnormalities between cases. Overall, 315 abnormalities have been identified (236 deletions and 79 gains), with a median of 4 copy-number abnormalities per patient (range 0-32). In patients with IgVH mutational status available the median number of chromosomal aberrations in the mutated group was 5 (range 1-17) and unmutated group was 4 (range 0-20). Copy number deletions were significantly more common than chromosomal gains for the total group and in both the unmutated and mutated IgVH gene groups. Mean number of megabases (Mb) of DNA deleted in the unmutated IgVH group was 17.80Mb (standard deviation 24.17 Mb, median 6.63 Mb, range 0 - 90.55Mb) and 40.65 Mb in the mutated IgVH group (standard deviation 86.04 Mb, median 15.35Mb, range 0 – 307.57Mb). Mean number of Mb gained in the unmutated group was 37.74Mb (standard deviation 57.58Mb, median 0.44Mb, range 0 – 132.47Mb) and 32.99Mb (standard deviation 59.76Mb, median 0.14Mb, range 0 – 172.50Mb) in the mutated group. Nine patients had B-CLL with VH1-69 genes and 8 of them were unmutated. Median number of chromosomal deletions was 2 and gains also 2 (ranges 0 – 8 deletions and 0 – 4 gains) for this group. Similar findings were seen for the other VH families. These results initially suggest that total copy number variations do not account for the observed clinical differences between mutated and unmutated IgVH B-CLL. The catalogue of potentially clinically relevant chromosomal abnormalities found in this aCGH study is being currently evaluated. Disclosures Zent: Genentech, Bayer, Genzyme, Novartis: Research Funding. Shanafelt:Genentech: Research Funding; Hospira: Membership on an entity's Board of Directors or advisory committees, Research Funding; Polyphenon E International: Research Funding; Celgene: Research Funding; Cephalon: Research Funding; Bayer Health Care Pharmaceuticals: Research Funding. Kay:Genentech, Celgene, Hospira, Polyphenon Pharma, Sanofi-Aventis: Research Funding; Biogenc-Idec, Celgene, Genentech, genmab: Membership on an entity's Board of Directors or advisory committees. Fonseca:BMS: Consultancy; Amgen: Consultancy; Otsuka: Consultancy; Celgene: Consultancy; Medtronic: Consultancy; Genzyme: Consultancy.

Abstract Abstract 2909 Poster Board II-885 Background: Previous studies have identified sole abnormalities of del(20q) and del(13q) as prognostically favorable cytogenetic markers in primary myelofibrosis (PMF) (Tefferi et al. BJH 2001;113:763). A more recent study (Tam et al. Blood 2009;113:4171) confirmed these findings and suggested additional cytogenetic markers of prognosis. In the current study with larger numbers of informative patients studied at time of diagnosis, we wanted to validate these observations and examine the prognostic interaction between cytogenetic risk categorization and the International Prognostic Scoring System (IPSS). Methods: Patients with cytogenetic information at diagnosis were selected from the Mayo Clinic database of WHO-defined PMF. Specific cytogenetic categories were considered only in the presence of at least 5 informative patients; otherwise, they were included in the category of “other cytogenetic abnormalities”. Follow-up information was updated in July, 2009. Survival curves were prepared by the Kaplan-Meier method and compared by the log-rank test. Cox regression model was used for multivariable analysis. Results: 200 patients were studied (median age, 62 years; 63% males). The IPSS risk distributions were low in 66 patients, intermediate (int)-1 in 64, int-2 in 44 and high in 26. Cytogenetic findings at diagnosis were abnormal in 83 (42%) patients and included sole del(20q) in 21, complex (i.e. 3 or more abnormalities) in 13, sole del(13q) in 8, sole +8 in 7, sole +9 in 6, and other abnormalities in 28 patients. Median survivals in low, int-1, int-2 and high IPSS risk groups were 188, 71, 47 and 26 months, respectively (p < 0.0001). Median survival in patients with sole +9 was not reached and in those with sole del(13q), sole del(20q), normal karyotype, complex abnormalities and sole +8 was 112, 108, 80, 37 and 27 months, respectively, while it was 46 months for patients with other cytogenetic abnormalities (p = 0.01). Accordingly, sole abnormalities of +9, del(20q) and del(13q) were categorized as being favorable (n = 35) and complex abnormalities and sole +8 as unfavorable (n = 20); the respective median survivals were 112 and 34 months (p=0.002; Figure). Multivariable analysis confirmed the IPSS-independent prognostic value of cytogenetic risk categorization and the intra-IPSS risk prognostic distinction was most apparent in the int-1 group: median survival was 35 and 81 months in the presence of unfavorable or favorable cytogenetic markers, respectively (p=0.0009; Figure). Conclusion: The current study identifies sole +9, along with del(13q) and del(20q), as favorable and sole +8, along with complex abnormalities, as unfavorable cytogenetic markers of prognosis in PMF. Cytogenetic risk categorization in PMF has an IPSS-independent prognostic value that is important in patient selection for specific therapy. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 1614 Poster Board I-640 Background Cytogenetic abnormalities in B-cell CLL are important prognostic indicators for predicting disease progression and treatment response. Until recently, only interphase cytogenetics has been readily applicable to clinical use in CLL due to the inability of traditional mitogens to promote effective metaphase cytogenetic identification. Recently, cytokine or CpG oligonucleotide stimulation has been utilized to promote efficient metaphase analysis. One concern with this approach is the actual clonality and reproducibility of such clones when examined by different cytogenetic laboratories. The CLL Research Consortium (CRC) conducted a prospective trial to test (1) whether CpG would enhance detection of abnormal clones and (2) whether the CRC cytogenetic laboratories would achieve similar results when testing the same patient samples. Methods Initially, one laboratory compared stimulation of CLL cells using CpG versus pokeweed mitogen (PWM) + 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in the same samples. To test the reproducibility of CpG stimulation, fresh blood samples from 1 normal control and 12 CLL patients were collected at one site and distributed (blinded to phenotype and prior cytogenetic results) to 5 CRC cytogenetic laboratories where they were cultured for 3 days using CpG stimulation, harvested, banded and analyzed utilizing standard laboratory procedures. When possible, each laboratory analyzed 20 metaphases per case. Interphase FISH analysis for CLL (including probes for chromosomes 6q, 11q, 12, 13q, 17p, and IGH/CCND1) was also done on each sample. Results In the PWM+TPA versus CpG comparison, more cases were found to have clonal abnormalities with CpG stimulation (147 of 229, 64%) than with PWM+TPA (110 of 229, 48%; P=0.0005). All clonal abnormalities detected in the PWM+TPA cultures were also observed in the CpG cultures. In the 5-laboratory CpG comparison, the results were compiled and compared to the concurrent FISH results. The CpG karyotypes were concordant with the FISH results. The control case had a normal CpG-stimulated karyotype in all 5 laboratories. Three cases exhibited a complex karyotype; one of these also exhibited 11q-, one had 17p-, and one had neither. The cytogenetic descriptions varied modestly among labs, but all identified the complex clones. Every case including the control exhibited some nonclonal abnormal cells, but no pattern was detected among the 5 labs. When an abnormal clone was found by only 1 or 2 labs, it was present in only a very small number of cells. A small 13q- clone was observed in 2 cases in 1 and 2 labs (3 of 20 and 5 of 50 cells, respectively), and was confirmed by interphase FISH. A single lab found 2 of 30 cells with a t(12;15) in one case. Conclusions Abnormal clones in CLL are more readily detected with CpG stimulation than with traditional B-cell mitogens. The clonal abnormalities revealed by CpG stimulation are consistent with the “gold standard” interphase FISH results, and are reproducible among different cytogenetic laboratories. We have no evidence that CpG oligonucleotides create new clonal B-cell populations in vitro during 3 days of cell culture. CpG stimulation in vitro reveals clonal cytogenetic abnormalities and complexity in CLL that should be evaluated as potential independent prognostic parameters. Disclosures Kay: Biogenc-Idec, Celgene, Genentech, genmab: Membership on an entity's Board of Directors or advisory committees; Genentech, Celgene, Hospira, Polyphenon Pharma, Sanofi-Aventis: Research Funding.

Updated WHO criteria define mixed phenotype acute leukemia (MPAL) with more stringent diagnostic criteria than the formerly described entity biphenotypic acute leukemia (BAL). The changes in diagnostic criteria influence management by assigning weight to aberrantly expressed markers and minimizing expression of myeloid markers other than myeloperoxidase (MPO), potentially foregoing consolidative allogeneic transplant for an otherwise “favorable” lymphoid phenotypic leukemia. We present a case of MPO-negative, myeloid antigen-positive acute lymphoblastic leukemia who progressed with refractory phenotypic acute myeloid leukemia while receiving lymphoid-directed therapy and discuss concerns raised by the adoption of the new, more stringent diagnostic criteria for BAL.

Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) is a low-grade B-cell neoplasm and ∼2% to 9% patients develop an aggressive lymphoma, most commonly diffuse large B-cell lymphoma (Richter transformation, DLBCL-RT). Programmed death-1 (PD-1) pathway plays a crucial role in tumor host immunity evasion and its blockade has emerged as an effective anti-cancer immunotherapy. PD-L1 and PD-1 expression has shown predictive value in anti-PD cancer immunotherapy; however, it has not been well documented in CLL/SLL and DLBCL-RT. We evaluated PD-1 and PD-L1 expression by immunohistochemistry in 39 CLL/SLL, 15 DLBCL-RT, and 26 other DLBCL. In CLL/SLL, neoplastic B-cell PD-1 expression was weak and restricted to prolymphocytes/paraimmunoblasts within proliferation centers (PCs) and accentuated PCs of all sizes. Neoplastic B-cell PD-1 expression was highly prevalent and demonstrated increased intensity in DLBCL-RT, but in contrast was only rarely seen in other DLBCL (12/15 vs. 1/26; P<0.0001). An excellent correlation (90% concordance) was observed between neoplastic B-cell PD-1 immunohistochemistry positivity and molecularly defined CLL/SLL clonal relatedness in DLBCL-RT. PD-L1 expression was observed on the neoplastic B cells in rare DLBCL-RT and other DLBCL cases (1/15 vs. 1/26; P>0.05) as well as background histiocytes and dendritic cells. Overall survival of DLBCL-RT was significantly inferior to that of the other DLBCL (median, 16.9 vs. 106.1 mo; P=0.002). Our findings suggest a biological continuum from prolymphocytes/paraimmunoblasts in CLL/SLL PCs to the neoplastic B-cells in DLBCL-RT. The characteristic PD-1 expression in DLBCL-RT makes it a potential surrogate marker for determining clonal relatedness to CLL/SLL, which may have important prognostic and therapeutic implications.