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Abstract Background: Castleman Disease (angiofollicular hyperplasia) is a rare, lymphoproliferative disease. There has been no large study describing the natural history of this disease. Hypothesis: A retrospective analysis of clinical, pathologic, and laboratory factors predictive of outcome should be identifiable. Methods: During the period from 3/30/48 to 6/18/2002, 114 patients with Castleman Disease were seen at the Mayo Clinic (Rochester, 56; Jacksonville, 19; Arizona, 13) and at the University of Nebraska (26). Pathology, laboratory and clinical characteristics analyzed included: the presence of unicentric or multicentric disease, the pathologic variant (hyaline vascular versus plasma cell variant), co-existing POEMS syndrome, age, serum albumin, alkaline phosphatase, AST, sedimentation rate, the presence of cytopenias, organomegaly (hepatomegaly or splenomegaly), papilledema, peripheral neuropathy, sclerotic bone lesions, renal disease, B-symptoms or respiratory symptoms. The impact of these variables on overall survival from time of diagnosis was evaluated using univariate analyses, where their significance was determined based on the logrank statistic. To derive the multivariable model, the score-based model-building method was used. Based on this approach, a 3-variable model was designed, adjusting for age at diagnosis. Patients were then assigned a risk score corresponding to how many of these risk factors they possessed. A final proportional hazards model was constructed based on this risk score. Results: The median age at diagnosis was 43 (range: 5 – 78), and 48% of patients were male. Fifty-five patients had multicentric disease. Median follow-up of living patients was 5.8 years (range: 0.02 – 27). On univariate analysis, factors that predicted for overall survival included: age at diagnosis, presence of multicentric disease, presence of a monoclonal protein in the urine, co-existing POEMS syndrome, serum albumin, presence of cytopenias, organomegaly, neuropathy, thrombocytosis, and respiratory symptoms. The final multivariable model included 98 patients. Adjusting for age, the model included organomegaly, respiratory symptoms, and thrombocytosis. Patients were assigned 1 point for each risk factor they possessed based on clinical relevance and similarity of the hazard ratios for each of these 3 variables (ranging from 3.1 to 4). A final prognostic survival model was constructed using this assigned risk score. These scores were collapsed further based on observed similarities (0 factors vs. 1 or more factors). This final model yielded a hazards ratio of 5.5 (95% CI 2.3–13.4). The 10 year survival rates were 80% (95% CI: 65–98%) and 41% (95% CI: 28–59%) for the 0 factor and 1+ factor groups, respectively. The 20 year survival rates were 71% (95% CI: 52–87%) and 31% (95% CI: 19–51%), respectively. Conclusion: The most distinguishing prognostic clinical, laboratory, and pathological characteristics of Castleman’s Disease are orgnaomegaly, respiratory symptoms, and thrombocytosis. This prognostic scoring system should aid in individual cases and in assessing results of therapeutic interventions.

Abstract INTRODUCTION: Previous studies have demonstrated the clinical activity of the mTOR inhibitor RAD001 in low- grade lymphomas. Our preclinical studies demonstrated activity of mTOR inhibitors in Waldenstrom Macroglobulinemia (WM) cell lines and patient samples. This phase II study aimed to determine safety and activity of the oral mTOR inhibitor RAD001 (Novartis Pharmaceutical, MA) in patients with relapsed or refractory WM. METHODS: Patients who had at least one previous therapy for WM, and who had symptomatic relapsed or refractory disease were eligible. NCI CTCAE v3.0 was used for toxicity assessment. All patients received daily RAD001 at 10 mg. A cycle was considered 28 days. Patients were allowed to stay on therapy until progression of disease or excessive toxicity. This study was conducted in a collaborative effort between Dana Farber Cancer Institute (DFCI) and Mayo Clinic College of Medicine. Here, we report the data on the patients accrued at DFCI. RESULTS: 19 pts (15 men and 4 women) have been treated to date. All patients had symptomatic disease and required therapy. The median number of lines of prior treatment was 3 (range 1 – 5) including included rituximab, nucleoside analogues (fludarabine or 2-CDA), combination chemotherapy (e.g. CHOP, CVP), chloramucil, and bortezomib. The median IgM at baseline was 3330 mg/dL (range 1010– 7410). The median follow on RAD001 was 8 months (range 3 – 22 months). Eighteen pts are currently evaluable for response. Best responses to RAD001 after 2 cycles using IgM monoclonal protein were as follows: partial remission in 8 (44%), minimal response in 5 (28%). Progressive disease occurred in 4 (22%) and stable disease occurred in 1 (6%). The overall response rate (PR+MR) was 72%. The median duration of response has not been reached (3–22+ months). Patients tolerated therapy well without significant toxicities. Grade 3 and 4 toxicities included grade 4 thrombocytopenia in 1 patient, grade 3 pneumonia in 1 patient, grade 3 hyperglycemia in 1 patient and grade 3 mucositis in 1 patient. Other adverse events of grade 2 or lower included nail cracking, mucositis, diarrhea, and fatigue. Attributable toxicities otherwise proved manageable with appropriate supportive care, and RAD001 was generally well tolerated. One patient enrolled on the study withdrew consent and changed to hospice care within 3 weeks of therapy, and passed away due to disease progression. CONCLUSIONS: The use of the oral RAD001 single agent RAD001 in patients with relapsed or refractory WM was welltolerated and demonstrated significant activity achieving an overall response rate in 72% of patients. Future studies of combination of this agent with rituximab and bortezomib are currently being planned.

Abstract On intergroup phase III trial, MRC UKALLXII/ECOG2993, 25% of 613 ECOG patients were positive for Ph or its molecular equivalent, BCR/ABL; all were B-lineage ALLs (B-ALL). When compared to Ph(−) B-ALL, Ph(+) ALL had more blasts expressing CD25 (α-chain of interleukin-2 [IL-2] receptor) (p=3E-31), CD34 (p=4E-10), CD13 (p=3.9E-16) and CD33 (p=3.5E-09), and blasts with higher density of CD10 (p=9.6E-12). There was no difference in antigen expression profiles between pts with e1a2 (N=107) and b2a2/b3a2 (N=48) BCR/ABL transcripts. Complete remission (CR) rate was lower (p= 7.4E-6) and overall survival (OS) was shorter in Ph(+) compared with Ph(−) B-ALL (p=6.5E-5). There was no difference in outcome between e1a2 and b2a2/b3a2 pts (p=0.65). CD25 was the most powerful prognostic indicator in Ph(+) cases, both for CR (p=0.004) and OS (p=1.3E-05). Strikingly, CD25 negatively affected OS in a continuous fashion; 1% increase in CD25 was related to a 1% increase in the hazard of death. On the other hand, CD25 expression was not associated with OS in Ph(−) pts (p=0.66). Among 152 Ph(+) pts, 30 lacked CD25 expression, 24 with e1a2 and 6 with b2a2/b3a2 transcripts. When analyzed separately, CD25(−)Ph(+) pts had outcome indistinguishable from that of Ph(−) pts (p=0.89). Compared to CD25(+)Ph(+) pts, CD25(−) Ph(+) pts had lower WBC counts at presentation (p=4.1E-05). Both CD25(+) and CD25(−) Ph(+) blasts expressed the γ-chain but not the β-chain of the IL-2 receptor, arguing against the presence of a functional IL-2 receptor. There was no association between CD25 expression or outcome and the presence of additional cytogenetic abnormalities seen in 52% of Ph(+) pts. To further examine the prognostic impact of CD25, BCR/ABL levels were quantitated by real-time PCR in 136 Ph(+) pts at presentation. There was no correlation between presenting BCR/ABL levels and blast count (p=0.98), achievement of CR (p=0.73), or OS (p=0.67). However, higher blast counts at diagnosis were associated with shorter survival (p=0.003). BCR/ABL levels were measured in 43 pts 1 month after induction therapy. Changes in BCR/ABL after therapy were classified as undetectable, >90% reduction, >50% reduction, or no change. While CR status did not correlate with BCR/ABL reduction (p=0.97), the chance of survival ranked in the order of undetectable (p=0.01), >90% reduction (p=0.05), >50% reduction (p=0.37), or no change (p=0.87). CD25 expression at presentation correlated with that order (p=0.002). CD25(−)Ph(+) pts fell into the group with undetectable BCR/ABL levels after treatment, whereas pts with highest initial CD25 expression fell into the group with no change. In conclusion, CD25(−)Ph(+) blasts are more sensitive to non-TKI therapy than CD25(+) Ph(+) blasts. Outcome in Ph(+) ALL is thus determined by CD25 expression rather than the presence of Ph per se. RESPONSE CD25(+)Ph(+) CD25(−)Ph(+) Ph(−) B-Lin ALL n 122 30 308 % CR Rate 57% 71% 81% MedianOS (months) 12.2 30.2 27.7

Abstract Over the past decade several prospective studies of post-remission therapy in AML have compared allogeneic hematopoietic stem cell transplantation (HSCT), autologous HSCT and chemotherapy. The data are appropriately presented by intention-to-treat analyses. However, such analyses do not provide information to the clinician or patient as to outcome from the initiation of post-remission therapy, be it HSCT or chemotherapy. Furthermore, such analyses are impossible to interpret when a significant percentage of patients do not receive their intended therapies (for example, only 54% of patients randomized to autologous HSCT; Cassileth, NEJM, 1998). Finally, such studies present survival data that may not be relevant to current practice where the mortality from autologous HSCT has decreased sharply (for example, 14% non-relapse mortality from autologous HSCT; Cassileth, NEJM, 1998). In the current analysis, results from 4 consecutive ECOG studies that included autologous HSCT were analyzed and the data collated. In PC486 patients received standard induction followed by allogeneic HSCT if they had a compatible sibling; otherwise they were to undergo an autologous HSCT without any prior consolidation (Cassileth, JCO, 1993). E4995 studied standard induction with added high-dose cytarabine (HiDAC) intensification on D 8–10, followed by 2 courses of HiDAC consolidation. All patients were to undergo an autologous HSCT after induction and consolidation (Cassileth, Leuk Lymph, 2005). E3489 was a US Intergroup study in which patients received standard induction (3 + 7). If in CR, all patients received an attenuated course of induction (2 + 5). Patients who had a histocompatible sibling were assigned to allogeneic HSCT; those without a donor were randomized to an autologous HSCT or chemotherapy (Cassileth, NEJM, 1998). E1900 is an ongoing phase III study in which patients receive standard induction followed by allogeneic HSCT or two courses of HiDAC followed by autologous HSCT. The latter study is ongoing; therefore, only data describing transplant-related mortality (TRM) are presented. The relapse rate from start of each post-remission regimen in these studies, based on therapy actually received and not on intention-to-treat analysis, is presented in table 1 and the TRM is presented in table 2. Table 1. Relapse Rate from Start of Post-Remission Therapy Cytogenetics Allogeneic HSCT (n=85) Autologous HSCT (n=113) Chemotherapy (n=109) Favorable 11.8% (2/17) 10.5% (2/19) 60% (12/20) Intermediate 20.5% (8/39) 32.3% (10/31) 53.7% (22/41) Unfavorable 16.7% (2/12) 41.7% (5/12) 83.3% (15/18) Unknown 23.5% (4/17) 33.3% (17/51) 56.7% (17/30) Table 2. Treatment-Related Mortality after Post-Remission Therapy Allogeneic HSCT Autologous HSCT – Marrow Autologous HSCT – Blood Chemotherapy Study E3489, E4995, E1900 E3489, PC 486 E4995, E1900 E3489 Treatment-related mortality 15.3% (18/117) 11% (16/85) 1.5% (2/135) 2.7% (3/109) In conclusion: The TRM after autologous HSCT using mobilized peripheral blood has decreased substantially in recent years. As shown in table 2, in PC486 and E3489 where bone marrow was harvested and treated with perfosfamide (4-HC) prior to cryo-preservation, the TRM was significantly greater than in the later studies, E4995 and E1900, where peripheral blood was harvested after priming with chemotherapy and/or cytokines. The lower relapse rate post autologous transplant, together with the lower procedural mortality with current practice, may confer a survival advantage in future prospective studies of autologous HSCT versus chemotherapy. While absolute comparisons cannot be made, as the timing of each therapy may not be identical, these data provide crucial information for informed and patient/physician discussions prior to initiation of postremission therapy.

Abstract Background: The combination of lenalidomide and dexamethasone (Rev/Dex) has been shown to be a highly effective therapy for multiple myeloma (MM). Over half of the patients with newly diagnosed MM achieve a complete response with prolonged therapy. More recently, a phase III trial demonstrated improved survival with use of lenalidomide and low dose dexamethasone with one year survival in excess of 90%. We report the initial results from a phase II trial combining lenalidomide and low dose dexamethasone with cyclophosphamide (CRd) as initial therapy of newly diagnosed MM. Methods: The trial was initiated in July 2006 and completed the target accrual of 33 patients by July 2007. The treatment protocol consisted of lenalidomide given orally at a dose of 25 mg daily on days 1–21 of a 28-day cycle. Dexamethasone (dex) was given orally at a dose of 40 mg on days 1, 8, 15, and 22 of each cycle. Cyclophosphamide at a dose of 300 mg/m2 was given on days 1, 8, and 15 of each cycle. Patients also received an aspirin once daily as thromboprophylaxis. Response was defined as a decrease in serum monoclonal (M) protein by 50% or higher and a decrease in urine M protein by at least 90% or to a level less than 200 mg/24 hours, confirmed at least 4 weeks apart. Results: The median age was 63 years (range, 44–79). Eight patients (24%) had ISS stage III disease. At this time, 19 of the 33 patients are evaluable for confirmed responses (i.e., off-study or completed at least 4 cycles of therapy). Of these, 2 achieved VGPR and 13 had a partial response giving an overall response rate of 79%. The response rate was affected by 5 of 19 patients who went off study within three cycles due to toxicities [interstitial nephritis (1 pt), multiple grade 3 toxicities including infection (1 pt) atrial fibrillation and infection (1 pt)] or alternative treatment [no response and possible renal toxicity (1 pt) and progression at 4 cycles (1 pt)]. Overall, hematological toxicity was the most common with grade 4 toxicity seen in 6 patients (20%). Non-hematological grade 3 or higher toxicities included fatigue (4 pts), thrombosis (3 pts) and renal failure (2 pts). One patient with thrombosis came off study for other toxicities, and the other two continued on study with anticoagulation. Thirteen patients (43%) had dose reductions of both cyclophosphamide and lenalidomide, most commonly due to hematological toxicity. So far, 12 patients have gone off study, 6 went to stem cell transplant, 3 due to adverse events, 1 due to disease progression and 2 patients went to alternate treatment. Conclusions: CRd has excellent activity in newly diagnosed myeloma with an estimated overall response rate of 79%. Response evaluation of the entire 33 patients will be presented at the meeting. We believe that the initial use of a 300 mg/m2 dose of cyclophosphamide resulted in 5 of the first 19 pts experiencing early toxicity and withdrawal. As a result, an expansion of the current trial is evaluating lower doses of cyclophosphamide (300 mg fixed dose). Full analysis of this study will be needed to determine if CRd can improve outcome compared to Rd.

Abstract MRC UKALLXII/ECOG2993 accrued 739 ECOG pts over 13 years. ECOG’s reference laboratories centrally characterized 613 (91%) and 505 were B-Lin ALLs. Expression of 32 antigens (Ags), including 9 myeloid-associated Ags, was determined by multiparameter flow cytometry on gated blasts, both as percentage of Ag expressing blasts and intensity of antibody staining (equivalent of Ag density). To test for the potential therapeutic efficacy of monoclonal antibodies, such as rituximab, epratuzumab, alemtuzumab, or gemtuzumab in future trials, expression of Ags for these antibodies (CD20, CD22, CD52, or CD33, respectively) was determined. In E2993, 14% of pts typed as Pro-B (CD10 negative), 71% as Early Pre-B (CD10 positive), 13% as Pre-B (intracytoplasmic mu positive), and 2% as Mature B-ALL (surface mu chains positive). CD20 expression correlated with the stage of B-lymphoid maturation (p=5.4E-13) (see table). Median CD20 intensity of fluorescence was lowest in Pro-B and highest in Mature B-ALL. Membrane expression of CD22 was lower in Pro-B compared with the other three groups, Early Pre-B, Pre-B and Mature B (p=1.0E-5) but indistinguishable among the latter three (p=0.21). While levels of CD20 and CD22 had no effect on complete remission (CR) and overall survival (OS) univariatly, higher CD22 expression was significantly correlated with better OS (p=0.02) in a bivariate Cox model of CD20 and CD22. With respect to myeloid Ags, CD33/CD13 expression was associated with Early Pre-B (p=0.02), and CD65(s)/CD15(s) with Pro-B ALL (p<0.0001). While CD65(s) and CD15(s) did not affect outcome, the combined expression of CD33/ CD13 conferred an inferior prognosis; this effect was due to the association of CD33/ CD13 with BCR/ABL positivity (p<0.0001). BCR/ABL negative B-Lin ALL (N=350) expressed CD33 on a median of only 4% of blasts, compared with a median of 54% in BCR/ABL positive ALL (N=152) (p=3.5E-09). However, intensity of CD33 staining was significantly lower in both BCR/ABL negative and positive blasts (median 3.1) compared with leukemic myeloblasts (median 56) (p=<0.001). Median CD52 expression was higher in BCR/ABL positive versus negative B-Lin ALL (p=3.5E-06); in neither group was CD52 associated with outcome. We conclude that antibodies to CD52 are better suited for BCR/ ABL positive than negative B-Lin ALL. Since expression of CD33 in ALL is weak, CD33- independent effects of gemtuzumab must be investigated. Low CD20 combined with high CD22 expression confers superior OS. The expression of these Ags in most B-Lin ALL pts justifies the incorporation of antibodies into frontline chemotherapy regimens. B-Lin ALL Stage CD20 Percentage Median Intensity CD22 Percentage Median Intensity ≥20% ≥50% ≥75% ≥20% ≥50% ≥75% Pro-B 21 10 3 2 88 72 55 12 Early PreB 66 48 37 21 96 90 84 28 Pre-B 61 46 37 21 100 90 81 37 Mature B 87 80 64 106 92 84 80 9

Abstract Background: Mantle cell lymphoma (MCL) is an incurable non-Hodgkin lymphoma. MCL is characterized by the chromosomal translocation t(11;14) leading to overexpression of cyclin D1 (CCND1), a regulator of the cell cycle. NF-κB plays a central role in key cellular processes, including cell cycle regulation and apoptosis, and it has been shown to be constitutively activated in MCL cell lines and in patient biopsy samples and to play a key role in the growth and survival of MCL cells. In an exploratory study, we evaluated the hypothesis that germline variation in candidate genes from the cell cycle and NF-κB pathways predict overall survival in MCL. Methods: We genotyped 235 SNPs from 29 cell cycle genes and 447 SNPs from 55 NF-κB genes in 39 MCL patients aged 38–64 years who participated in a population-based study conducted from 1998–2000 through the Surveillance, Epidemiology, and End Results (SEER) cancer registries in Detroit, Seattle, Iowa, and Los Angeles county. Tagging single nucleotide polymorphisms (SNPs) were selected from HapMap. Stage, presence of B-symptoms, first course of therapy, date of last follow-up, and vital status were derived from linkage to registry databases at each study site. To assess the statistical significance of a gene, we first used principal components analysis to capture the genetic variation of the SNPs within a gene. Cox proportional hazards analysis was then used to assess the association between the top principal components from a gene with overall survival. Results: Through early 2005, there were 22 deaths in 39 patients (56%). The median follow-up of the 17 surviving patients was 56 months (range 36–70). Eleven of the 55 genes (TNFSF14 p=0.0007, NF-κBIA p=0.003, IL1R2 p=0.01, TNFRSF25 p=0.02, TNFSF13B p=0.02, FAS p=0.03, TLR2 p=0.04, TNFSF10D p=0.04, TNFSF10 p=0.06, MAP3K2 p=0.06) in the NF-κB pathway were associated with overall survival. In the cell cycle pathway, only 3 of the 29 genes (CASP5 p=0.06, CASP9 p=0.09, BCL2 p=0.09) were associated with overall survival, and a previously reported SNP in CCND1 (rs603965) showed no association (p=0.5). Permutation p-values for observing at least this number of associated tests in each pathway due to chance (from multiple testing) was p=0.8 for the cell cycle pathway and p=0.03 for NF-κB pathway. The small sample size precluded multi-gene modeling. Conclusion: In a population series of MCL patients, we found suggestive evidence that host genetic variation in candidate NF-κB pathway genes affects overall survival. These results complement tumor expression data implicating this pathway in the pathogenesis of MCL. In addition, such findings further support the value of developing therapeutic targets from this pathway.

Abstract Results from clinical trials may change with time. Data presented in initial abstracts or early reports are often different from what appears in subsequent publications, sometimes due to a more rigorous analysis (as in initial response rates) or to maturity of data (as in reports of survival). Survival curves usually provide projected data based on Kaplan-Meier analyses. Such data may change with time when unexpected events occur (as, for example, relapse after T-cell depleted allogeneic transplants) or more patients advance to the initially projected time point. For AML, several groups have reported that there are very few relapses beyond 3 years. At issue is the optimal time for reporting these survival data. Table 1 reports data from 6 consecutive clinical trials addressing induction and post-remission questions in AML patients performed by ECOG. These data compare the 3-year overall survival (OS) and disease-free survival (DFS) for the identical cohorts of patients as presented at 1 year and at 3 years after completion of the study accrual. PC 486 was a phase II study of induction therapy followed by allogeneic or autologous transplant without prior consolidation. E 3489 was a US intergroup study in which patients received post-induction therapy using an allogeneic transplant (allo), an autologous transplant (auto) or chemotherapy (chemo). E1490 was a study in older adults of induction and consolidation with GM-CSF or placebo. E2491 was an intergroup study comparing ATRA induction versus standard chemotherapy in patients with APL. After consolidation, patients were randomized to maintenance with ATRA versus observation (obs). E3993 was a study in older adults comparing priming with GMCSF or placebo. E4995 was a phase II study of intensified induction and consolidation followed by an autologous transplant. For the same cohorts of patients the 3-year OS tends to decrease as the time from completion of study accrual increases. When looking at the DFS, while a decrease may also occur, in several studies there was an improved DFS with time (for example, in E2491, chemo to ATRA, or in E3993 with GM-CSF priming). There were no changes in either OS or DFS beyond 3 years (data not shown). Table 1: Three-year OS and DFS Presented at Different Time Points OS (%) DFS (%) Study One year Three years One year Three years PC 486 auto or allo HSCT 42 → 38 45 → 39 E 3489 allo HSCT 50 → 50 48 → 47 auto HSCT 47 → 46 39 → 39 chemo 56 → 51 36 → 36 E 1490 Induction / consolidation GM-CSF 23 → 22 23 → 24 placebo 15 → 12 16 → 15 E2491 APL induction+ maintenance ATRA + ATRA 87 → 84 72 → 73 ATRA + obs 83 → 80 61 → 60 chemo + ATRA 81 → 75 43 → 58 chemo + obs 54 → 45 16 → 18 E 3993 Induction/consolidation GM-CSF priming 13 → 11 5 → 10 placebo 14 → 14 9 → 9 E 4995 Induction / consolidation / auto HSCT 54 → 48 63 → 53 In conclusion: For large cooperative group studies, treatment results in AML presented one year from conclusion of study accrual are unlikely to dramatically alter the overall results. Nonetheless, some differences may occur, both in OS and DFS, warranting caution in interpreting the data and comparing with other published reports. Such differences are likely to be even greater if data are presented upon completion of study accrual, prior to one year - although, in a cooperative group setting, this should be balanced against the need to release data for future planning. Publication of interim data, before conclusion of the study accrual, may be misleading and are to be strongly discouraged. Three years appear to be the appropriate time for the publication of definitive survival data.

Abstract We previously demonstrated that apoptosis-inhibiting proteins (e.g., Bcl-2, Mcl-1, and XIAP) are important in predicting response to chemotherapy and antibody therapy for CLL. Additionally, genetic abnormalities and epigenetic control of gene expression have become increasingly important in understanding the prognosis and potential therapeutic opportunities in this disease. Greater than 80% of patients with CLL have genetic abnormalities detectable by FISH. Important subsets include those with a less favorable prognosis (deletions of 17p, 11q), an intermediate prognosis (trisomy 12), or a favorable outcome (deletion of 13q). PTPRO, encoding a receptor-type tyrosine phosphatase, is a specific tumor suppressor gene located in chromosomal region 12p12.3. PTPRO has been highly characterized as inhibiting anchorage-independent tumor growth in human lung carcinomas. This gene has been found to be silenced by promoter methylation, and can be re-expressed by hypomethylating agents in human lung tumor models. Thus, we examined the methylation profile of the promoter region of PTPRO in primary leukemic cells from patients with CLL, and in B cells obtained from normal volunteers. Analysis of the PTPRO CpG island by methylation-specific PCR and combined bisulfite restriction analysis (COBRA) demonstrated that this region was methylated in 91% of samples from 46 patients, whereas it was unmethylated in control B cells from normal volunteers. Specific quantitative determinations of important proteins related to apoptosis in the leukemic cells from 20 of these patients showed that, in the group with methylation of the PTPRO promoter, there was a significant elevation of Bcl-2 (p=0.079), Mcl-1 (p=0.004), XIAP (p=0.048), Bcl-2/BAX ratio (p=0.0024), and Mcl-1/BAX ratio (p=0.0017). There was no apparent relationship with FISH category or immunoglobulin VH mutational status. Thus, PTPRO promoter methylation analysis may identify a subset of CLL patients less likely to respond to therapy, and provide a potential reversible target for new therapeutic approaches to this disease. Finally, analysis of methylation of CLL samples by restriction landmark genome scanning also revealed methylation of genes for three additional protein tyrosine phosphatases: PTPRN2, PTPRZ2, and PTPN11. Increases in methylation of two of these (PTPRN2 and PTPRZ2) in CLL samples relative to control B cells was confirmed by COBRA. These results provide additional potential molecular targets for CLL therapy.

Abstract Background: Previous cytogenetic studies in polycythemia vera (PV) have included a relatively small number of patients (“n” ranging 10–64). In the current study (n=137), we describe cytogenetic findings at presentation and examine their relationship to clinical and laboratory features, including bone marrow JAK2V617F allele burden. Methods: The study consisted of a consecutive group of patients with PV who fulfilled the World Health Organization (WHO) diagnostic criteria and in whom bone marrow biopsy and cytogenetic studies were performed at diagnosis. Results I: cytogenetic details At diagnosis: A total of 137 patients (median age, 64 years; 49% females) were studied at diagnosis and had adequate metaphases for interpretation. Cytogenetics were normal in 117 patients (85%) and displayed either a sole -Y abnormality in 5 patients (7% of the male patients), and other chromosomal abnormalities in 15 (11%). The latter included trisomy 8 in five patients, trisomy 9 in three patients, two patients each with del(13q), del(20q), and abnormalities of chromosome 1, and one patient each with del(3)(p13p21), dup(13)(q12q14), and del(11)(q21). At follow-up: Repeat cytogenetic studies while still in the chronic phase of the disease were performed in 19 patients at a median of 60 months (range, 8–198) from diagnosis. Of these, 4 had aquired new cytogenetic clones including 3 with normal cytogenetics at time of initial PV diagnosis. The new abnormalities included del(20q), del(5q), del(1p), chromosome 1 abnormality, and inv(3)(q21q26.2). At time of disease transformation: Leukemic transformation was documented in 3 patients of whom cytogenetic information at the time was available in 2 patients; both patients had normal results at time of initial PV diagnosis and complex cytogenetic abnormalities at time of leukemic transformation. In contrast, among 6 patients with available cytogenetic information at time of fibrotic transformation, the results were unchanged from those obtained at time of diagnosis in 5 patients. ii) Correlation between cytogenetics at diagnosis and JAK2V617F allele burden: Allele-specific, quantitative PCR analysis for JAK2V617F was performed in 71 patients using genomic DNA from archived bone marrow obtained at the time of the initial cytogenetic studies. JAK2V617F mutation was detected in 64 of the 71 (90%) patients; median mutant allele burden was 16% (range 3–80%) without significant difference among the different cytogenetic groups: normal vs. –Y vs. other cytogenetic abnormalities (p=0.72). iii) Clinical correlates and prognostic relevance of cytogenetic findings at diagnosis: Among several parameters studied for significant correlations with cytogenetic findings at diagnosis, an association was evident only for age (p=0.02); all –Y abnormalities (n=5) as well as 13 of the 15 (87%) other cytogenetic abnormalities occurred in patients ≥ 60 years of age. Stated another way, the incidence of abnormal cytogenetics (other than -Y) was 4% for patients younger than age 60 years and 15% otherwise. The presence of abnormal cytogenetics at diagnosis had no significant impact on either overall or leukemia-free survival. Conclusions: Abnormal cytogenetic findings at diagnosis are infrequent in PV, especially in patients below age 60 years. Furthermore, their clinical relevance is limited and there is not significant correlation with bone marrow JAK2V617F allele burden.