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Progression of chronic myelogenous leukemia (CML) to accelerated (AP) and blast phase (BP) is because of secondary molecular events, as well as additional cytogenetic abnormalities. On the basis of the detection of JAK2, CBL, CBLB, TET2, ASXL1, and IDH1/2 mutations in myelodysplastic/myeloproliferative neoplasms, we hypothesized that they may also contribute to progression in CML. We screened these genes for mutations in 54 cases with CML (14 with chronic phase, 14 with AP, 20 with myeloid, and 6 with nonmyeloid BP). We identified 1 CBLB and 2 TET2 mutations in AP, and 1 CBL, 1 CBLB, 4 TET2, 2 ASXL1, and 2 IDH family mutations in myeloid BP. However, none of these mutations were found in chronic phase. No cases with JAK2V617F mutations were found. In 2 cases, TET2 mutations were found concomitant with CBLB mutations. By single nucleotide polymorphism arrays, uniparental disomy on chromosome 5q, 8q, 11p, and 17p was found in AP and BP but not involving 4q24 (TET2) or 11q23 (CBL). Microdeletions on chromosomes 17q11.2 and 21q22.12 involved tumor associated genes NF1 and RUNX1, respectively. Our results indicate that CBL family, TET2, ASXL1, and IDH family mutations and additional cryptic karyotypic abnormalities can occur in advanced phase CML.

Purpose Acquired somatic uniparental disomy (UPD) is commonly observed in myelodysplastic syndromes (MDS), myelodysplastic/myeloproliferative neoplasms (MDS/MPN), or secondary acute myelogenous leukemia (sAML) and may point toward genes harboring mutations. Recurrent UPD11q led to identification of homozygous mutations in c-Cbl, an E3 ubiquitin ligase involved in attenuation of proliferative signals transduced by activated receptor tyrosine kinases. We examined the role and frequency of Cbl gene family mutations in MPN and related conditions. Methods We applied high-density SNP-A karyotyping to identify loss of heterozygosity of 11q in 442 patients with MDS, MDS/MPN, MPN, sAML evolved from these conditions, and primary AML. We sequenced c-Cbl, Cbl-b, and Cbl-c in patients with or without corresponding UPD or deletions and correlated mutational status with clinical features and outcomes. Results We identified c-Cbl mutations in 5% and 9% of patients with chronic myelomonocytic leukemia (CMML) and sAML, and also in CML blast crisis and juvenile myelomonocytic leukemia (JMML). Most mutations were homozygous and affected c-Cbl; mutations in Cbl-b were also found in patients with similar clinical features. Patients with Cbl family mutations showed poor prognosis, with a median survival of 5 months. Pathomorphologic features included monocytosis, monocytoid blasts, aberrant expression of phosphoSTAT5, and c-kit overexpression. Serial studies showed acquisition of c-Cbl mutations during malignant evolution. Conclusion Mutations in the Cbl family RING finger domain or linker sequence constitute important pathogenic lesions associated with not only preleukemic CMML, JMML, and other MPN, but also progression to AML, suggesting that impairment of degradation of activated tyrosine kinases constitutes an important cancer mechanism.

Background T-cell large granular lymphocytic leukemia is a clonal proliferation of cytotoxic T-lymphocytes which often results in severe cytopenia. Current treatment options favor chronic immunosuppression. Alemtuzumab, a humanized monoclonal antibody against glycophosphatidylinositol-anchored CD52, is approved for patients refractory to therapy in other lymphoid malignancies. Design and Methods We retrospectively examined treatment outcomes in 59 patients with CD8+ T-cell large granular lymphocytic leukemia, 41 of whom required therapy. Eight patients with severe refractory cytopenia despite multiple treatment regimens had been treated with subcutaneous alemtuzumab as salvage therapy. Flow cytometry was used to monitor expression of glycophosphatidylinositol-anchored CD52, CD55, and CD59 as well as to characterize T-cell clonal expansions by T-cell receptor variable β-chain (Vβ) repertoire. Results Analysis of the effects of alemtuzumab revealed remissions with restoration of platelets in one of one patient, red blood cell transfusion independence in three of five patients and improvement of neutropenia in one of three, resulting in an overall response rate of 50% (4/8 patients). Clonal large granular lymphocytes exhibited decreased CD52 expression post-therapy in patients refractory to treatment. Samples of large granular lymphocytes collected prior to therapy also unexpectedly had a significant proportion of CD52-negative cells while a healthy control population had no such CD52 deficiency ( p =0.026). Conclusions While alemtuzumab may be highly effective in large granular lymphocytic leukemia, prospective serial monitoring for the presence of CD52-deficient clonal cytotoxic T-lymphocytes should be a component of clinical trials investigating the efficacy of this drug. CD52 deficiency may explain lack of response to alemtuzumab, and such therapy may confer a survival advantage to glycophosphatidylinositol-negative clonal cytotoxic T-lymphocytes.

Abstract 3396 Chronic myeloid leukemia (CML) is characterized by the BCR/ABL fusion gene. However, secondary molecular events leading to accelerated (AP) or blast phase (BP) have not been sufficiently clarified. We hypothesized that, in analogy to other MDS/MPN or MPN, TET2, ASXL1, CBL and IDH family mutations may also occur in CML and contribute as secondary events leading to progression to AP or BP. Similarly, higher resolution of cytogenetic testing by single nucleotide polymorphism array (SNP-A)-based karyotyping may reveal additional chromosomal abnormalities associated with stepwise progression. This study is focused on the combined analysis of chromosomal lesions and mutations associated with AP, BP and Philadelphia chromosome (Ph) positive acute lymphoblastic leukemia (ALL) and the association of these defects with clinical features. We screened TET2, ASXL1, CBL and IDH for mutations in AP (N=14) and BP (N=26) and Ph+ ALL (N=9). Chronic phase (CP) (N=14) and Ph negative ALL (N=9) served as controls. We identified 3 CBL family (9%), 7 TET2 (21%), 2 ASXL1 (6%) and 2 IDH family (6%) mutations in patients with AP and myeloid BP. Subsequently, we also detected a TET2 mutation in a case of Ph+ ALL. None of these mutations were found in patients with CP or Ph negative ALL. We also performed SNP-A-based karyotyping and only included lesions which did not overlap with copy number variations (CNVs) or germ line regions of homozygosity present in any of the controls. 23 gains, 21 losses and 4 regions of somatic UPD lesions were identified. By SNP-A, additional copy number abnormalities, including microdeletions were found in 67% and 50% of patients with AP and BP, respectively. Recurrent lesions were detected on chromosome 1, 8, 9, 17 and 22. Microdeletions on chromosome 17 and 21 involved tumor associated genes NF1 and RUNX1. Deletions flanking the ABL1 and BCR genes were observed in 3 cases with der(22)t(9;22) or der(9)t(9;22) by metaphase cytogenetics. Gains including 1q25.3q41, chromosome 8 and 17q24.3 were found in 3 cases. Regions of UPD included UPD5q, 8q, 11p and 17q but no UPD involving 11q (CBL) and 4q (TET2) regions were found confirming heterozygous nature of the corresponding mutations. Newly detected molecular lesions associated with AP and BP may change the biology and thereby clinical features of affected cases. Overall survival of patients with mutations did not differ from those without mutations. Of note is that BCR/ABL1 kinase domain mutations were detected in 9/10 patients with imatinib resistance. In these 9 cases, 3 TET2 and 2 CBLB mutations were detected (but no mutations in the other genes). In an imatinib-resistant patient without BCR/ABL1 kinase domain mutation, CBL mutation was present. In the patients with TET2 mutations, additional chromosomal lesions were found by SNP-A, significantly more frequently when compared with WT cases (P=0.017). Of the 9 TET2 variants in 8 cases, 7 (78%) were missense substitutions, 1 (11%) was frame shift and 1 (11%) produced a stop codon and were located within the N-terminus as well as in a conserved DSBH 2OG-Fe(II)-dependent dioxygenase domain. The presence of nonsense and frameshift mutations suggests that mutated lesions result in inactivation, consistent with putative tumor suppressor functions, while heterozygous mutations indicate that the wild type allele is not completely protective. Since no TET2 mutations were identified in chronic phase CML, these mutations might represent an additional pathogenic event and contribute to progression. In 3 cases we observed a combination of 2 mutations. Coincidence of CBLB and TET2 mutations in 2 cases suggests that these might cooperate in the evolution of advanced phase of CML. We also found a combination of IDH1 and ASXL1 mutations in a patient with BP, suggesting that both mutations contribute to clonal advantage. In conclusion, while CBL family, ASXL1 and IDH family mutations as well a additional unbalanced chromosomal abnormalities not seen by metaphase cytogenetics can occur in myeloid type advanced phase CML, TET2 mutations were identified in Ph+ ALL, as well as myeloid BP and AP. These mutations likely represent secondary lesions which contribute to either disease progression or more aggressive features and commonly occur in association with imatinib-resistant BCR/ABL mutations. Disclosures: No relevant conflicts of interest to declare.

Abstract 420 Juvenile myelomonocytic leukemia (JMML) is a distinct subtype of myelodysplastic syndrome/myeloproliferative disorder (MDS/MPD) which, in analogy to chronic myelomonocytic leukemia (CMML), is characterized by excessive proliferation of myelomonocytic cells, but unlike CMML it occurs in young children and shows characteristic hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF). Chromosomal defect are present in 22% of patients in particular involving del7/7q. Mutations of genes involved in GM-CSF signal transduction, including RAS and PTPN11, can be identified in a majority of children with JMML, constitutional mutations of NF1 can be found in another 10% of patients with JMML, but in significant proportion of patients no molecular lesions were identified. To further clarify the molecular pathogenesis of JMML we have applied a high density SNP array-based karyptyping and screened for associated mutations including established defected in RAS, PTPN11 and NF1 but also c-Cbl and TET genes, recently identified in patients with CMML and MDS/MPD. We studied 49 children with JMML diagnosed between 1988 and 2008 in 28 institutions throughout Japan. The median age at diagnosis was 28 months (range, 1-75 months). Karyotypic abnormalities were detected in 11 patients, including 7 patients with monosomy 7. Two children had clinical evidence of NF1 mutations. Out of 49 patients, 32 received hematopoietic stem cell transplantation (HSCT). We performed mutational analysis of the genes known to be affected by mutations in JMML. PTPN11 mutations were found in 26/49 (53%) while NRAS and KRAS mutations were found in 2/49 (4%) and 1/49 (2%), respectively. None of the patients screened showed the presence of TET2 mutations, previously shown to be present in a significant proportion of patients with MDS/MPD, including CMML. High-density Affymetrix 250K single nucleotide polymorphism array (SNP-A) were applied as a karyotyping platform to identify LOH and submicroscopic copy number changes. Signal intensity was analyzed and SNP calls determined using Gene Chip Genotyping Analysis Software Version 4.0 (GTYPE). Copy number and areas of UPD were investigated using Hidden Markov Model and CNAG v3.0 software. Compared to the results of conventional metaphase cytogenetics (MC), SNP-A identified significantly more genetic abnormalities (25% vs 49%; p=.02). In 1 patient UPD17q was present ivolving NF1 locus. In 4 patients UPD11q involving c-cbl locus (11q23.1) was found. Sequencing of c-Cbl gene family revealed mutations of c-Cbl in 5/49 (10%), and no Cbl-b mutations. All but 1 mutations were homozygous and were located in the RFD (exon 8 and intron 8). C-Cbl mutations were mutually exclusive with PTPN11, NRAS, and KRAS mutations or had clinical diagnosis of NF1. Unlike in CMML, no UPD4q24 or homo- or heterozygous TET2 mutations were found. Histomorphologic analysis did not reveal any distinct c-Cbl mutation-associated features or differences in count between patients grouped based on the presence of specific mutations. Similarly, there were no differences in gender, or the presence of cytogenetic abnormalities and the probability of 2 year overall survival of c-Cbl mutant cases between patients grouped according to mutational status. All patients with c-Cbl mutations displayed GM-CSF hypersensitivity at initial presentation but did not differ in this feature from the remaining JMML patients. However, mutant c-Cbl cases showed earlier presentation (median age 12 months vs. 29 months, p = .037) and lower median hemoglobin F fraction (3.5 % vs. 25%, p=.02). In sum, c-Cbl mutations constitute a novel important pathogenic lesion in JMML. While their presence suggests functional similarity to CMML, absence of TET2 mutation in JMML and rarity of PTPN11 mutations in CMML constitute important distinctive features of both diseases. Disclosures: No relevant conflicts of interest to declare.

Abstract 416 Chronic myelomonocytic leukemia (CMML) is characterized by monocyte/monoblast proliferation, dysplastic features, progression to leukemia and poor prognosis. With the exception of rare cases of CMML with balanced PDGFRa or PDGFRb translocations, the molecular pathogenesis of the majority of CMML cases remains elusive. To date, somatic mutations pathogenic for CMML have not been identified, and large tyrosine kinase sequencing projects have been negative. The recent application of SNP-A as a karyotyping tool has allowed for more precise analyses of chromosomal defects and detection of copy-neutral loss of heterozygosity (CN-LOH). Previously, various recurrent areas of CN-LOH have been found in myeloid malignancies, but these lesions were particularly frequent in CMML. We have examined 68 patients with CMML and AML with antecedent CMML for recurrent areas of LOH, mapped mutations in genes contained within corresponding minimally affected regions, and examined their impact on clinical outcomes. We focused on Cbl, TET2 and RAS mutations and the resultant clinical and pathomorphologic phenotypes and outcomes associated with individual mutations. SNP-A-based karyotyping showed the presence of chromosomal aberrations in a larger proportion of cases than did metaphase cytogenetics: recurrent areas of somatic UPD were found in 49% of patients, with the most common shared lesions including UPD4q (n=6), UPD7q (n=6) and UPD11q (n=4). Initial identification of recurrent UPD11q and UPD4q and micro-deletions defining the smallest commonly affected areas led to the discovery of new mutations in CMML, including c-Cbl and TET2. In sum, we identified c-Cbl in 13%, TET2 in 49%, and RAS mutations in 10% of patients with CMML and sAML derived from CMML. LOH involving chromosome 7 was present in 18% of patients. All c-Cbl mutations were somatic and in the RING finger domain or linker sequence; 3/6 mutations were homozygous and one case with del11q harbored a hemizygous mutation. TET2 alterations were identified in 23 patients; in 26% of patients, both alleles were affected. However, no clinical differences were found between heterozygous and homozygous cases of TET2 mutations, suggesting a dominant negative effect. We also identified patients who harbored both TET2 and c-Cbl or RAS mutations demonstrating a multi-step pathogenesis of CMML. In addition, aberrant methylation at the CpG islands of promoters of TET2 (2 sites), c-Cbl (2 sites), b-Cbl (2 sites) and RAS (7 sites) was found to be relatively infrequent among patients. Intricate analysis of clinical and phenotypic features did not reveal pathognomonic phenotypes; however TET2 and c-Cbl mutations were associated with higher WBC (p=.027) or thrombocytopenia (p=.026), respectively and RAS mutations were associated with advanced disease (p=.027). Patients with LOH7q were included in our analysis as a separate group because they likely carried mutations in a putative candidate gene on 7q. In general, megakaryocyte nuclei displayed aberrant STAT5 staining in 31% of CMML cases (n=32) and correlated with morphologic dysplasia. Aberrant pSTAT5 staining suggestive of downstream pathways involving proliferative signals was present in 43% of TET2 mutated patients and 67% of c-Cbl mutants. None of the RAS mutants displayed aberrant STAT5 staining. Thus, aberrant STAT5 activation is not an obligatory feature of TET2 and RAS mutant cases, as opposed to the majority of c-Cbl mutant cases. There was no significant difference in survival between patients with TET2, c-Cbl and RAS mutations or those with LOH7q as compared to those without. Nevertheless, TET2 and c-Cbl mutant cases trended towards less favorable survival within lower-risk ( In sum, our results characterize CMML as a disorder frequently associated with acquired chromosomal lesions, activated STAT5 signaling, genetic mutations in RAS, ubiquitin modification and TET2 pathways. Mutations present in these patients may help to clarify pathogenic pathways, and thereby develop directed therapies. Disclosures: No relevant conflicts of interest to declare.

Abstract 2173 Poster Board II-150 Loss of heterozygosity (LOH) due to acquired uniparental disomy (UPD) is a commonly observed chromosomal lesion in myeloproliferative neoplasms (MPN) and myelodysplastic/myeloproliferative neoplasms (MDS/MPN) including chronic myelomonocytic leukemia (CMML). Most recurrent areas of LOH point towards genes harboring mutations. For example, UPD11q23.3 and UPD4q24 were found to be associated with c-Cbl and TET2 mutations, respectively. Cbl family mutations (c-Cbl and Cbl-b) have been associated with atypical MDS/MPN including CMML and juvenile myelomonocytic leukemia (JMML) as well as more advanced forms of MDS and secondary AML (sAML). Ring finger mutants of Cbl abrogate ubiquitination and thereby tumor suppressor function related to inactivation of phosphorylated receptor tyrosine kinases, Src and other phosphoproteins. TET2 mutations are present in a similar disease spectrum. The TET family of proteins is involved in conversion of methylcytosine to methylhydroxycytosine which cannot be recognized by DNMT1. Thereby, the proteins seem to counteract maintenance hypermethylation. In our screen of MDS/MPN, we found c-Cbl and Cbl-b ring finger mutations in 5/58 (9%) of CMML and AML derived from CMML, 2/39 (5%) MDS/MPNu, 4/21 (19%) JMML and 14/148 (9%) RAEB/sAML. In the same cohort, TET2 mutations were present in 37% and 14% of patients with MDS/MPN and MDS, respectively. Of note we did not find any TET2 mutations in JMML. We and others have also noted that TET2 and c-Cbl mutations were also detected in atypical chronic myeloid leukemia. While translocations resulting in BCR/ABL fusion characterize CML, we stipulated that in analogy to other chronic myeloproliferative diseases, TET2 and c-Cbl mutations may be also present in CML and contribute to phenotypic heterogeneity within BCR/ABL associated disorders. In particular, progression of CML to accelerated phase (AP) or blast crisis (BC) could be associated with acquisition of additional lesions. When 22 patients with CML chronic phase (CP) were screened, no TET2 and c-Cbl mutations were found. However, we identified 1 c-Cbl, 2 Cbl-b (6%) and 6 TET2 (12%) mutations in 51 patients with CML-AP (N=18) and CML-BC (N=33) with myeloid and lymphoid/mix 24 and 9 phenotype, respectively. These mutations were mutually exclusive. We also noted that TET2 mutations were present in 1/9 CML in BC with lymphoid phenotype. We subsequently screened Ph+ ALL cases (N=9) and found a TET2 mutation in 1 case but no Cbl family mutations. In contrast when 9 Ph- ALL cases were screened as controls, neither TET2 or Cbl mutations were found. SNP-A analysis revealed 2 cases of LOH involving chromosome 4 (UPD4q24 and del4) in a patient with lymphoid blast crisis and Ph+ ALL, respectively. However, UPD was not found in Cbl family gene regions (11q23.3 or 3q13.11). A homozygous deletion of Cbl-b region was seen in a CP patient. Cbl family mutations were associated with a more complex karyotype than TET2 mutations (67% vs. 17% cases with abnormal phenotype). Patients with Cbl family mutations were resistant to imatinib which was effective in only 2 out of 6 patients with TET2mutations. Dasatinib was effective in 2 patients with TET2 mutation. Median over all survival of progressed CML was 47, 49 and 48 months in patients with Cbl, TET2 or no mutations, respectively. In conclusion, our results indicate that Cbl family mutations can occur as secondary lesions in myeloid type aggressive CML (AP and myeloid BC), but not in lymphoid types. TET2 mutations were identified in both lymphoid BC and Ph1+ALL, as well as myeloid BC and AP. In contrast to CMML or JMML in which a vast majority of mutations are homozygous, all Cbl family mutations were heterozygous (no LOH). Similarly, all but two TET2 mutations were heterozygous (1 hemizygous in del4 and 1 homozygous case in UPD4q), suggesting that additional cooperating lesions affecting corresponding pathways may be present. These mutations likely represent secondary lesions which contribute to more either progression (CML) or more aggressive features (Ph+ ALL) and characterize disease refractory to therapy with imatinib. Disclosures: No relevant conflicts of interest to declare.

Abstract 1606 Poster Board I-632 Aberrant epigenetic silencing of tumor suppressor and differentiation genes constitutes an important mechanism in the pathogenesis of MDS and related myeloid malignancies. Demethylationg agents such as azacitidine and decitabine lead to degradation of DNMT1 and may reverse aberrant methylation. While the drugs demonstrate efficacy in MDS, response rates are variable. Thus, many primarily refractory patients are exposed to these therapies unnecessarily. While search for markers of responsiveness included study of methylation status of potential marker promoters or global methylation patterns, to date predictive tests have not been developed. Similarly, mechanisms of epigenetic instability responsible for wide-spread promoter methylation have not been clarified, preventing development of diagnostic markers. TET2 mutations are frequent events in a variety of MDS subtypes, particular chronic myelomonocytic leukemia (CMML) and other MDS/MPN, as well as AML derived from those conditions. It is likely that TET2 alterations are important in the pathogenesis of myeloid malignancies, but little is known regarding the function of TET2. A recent report indicated that a related family member, TET1, converts 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (hmC). Hydroxylation of 5mC prevents DNMT1 from homologous methylation of daughter DNA strands during cell division, thus preventing maintenance methylation (Tahiliani et al. Nature 324, 2009). Consequently, closely related TET2 may play a role in epigenetic regulation. As a consequence, TET2 mutations may lead to accumulation of aberrantly methylated CpG islands. Of utmost importance is whether TET2 mutations or resultant epigenetic silencing of specific gene or gene groups affects response to hypomethylating agents. We hypothesized that TET2 mutations play a role in epigenetic instability and may serve as markers of responsiveness/refractoriness to the therapy with demethylating agents. We have determined TET2 mutational status by sequencing all exons in 32 patients with myeloid malignancies (MDS (N=18) and MDS/MPN (N=14)) who underwent therapy with the demethylating agents azacitidine (N=27) or decitabine (N=5). For definition of response we applied International Working Group Criteria in patients who received a sufficient dose and number of cycles to allow assessment of response. Overall response rate (complete+partial responses (CR+PR) + hematologic improvement (HI)) was achieved for 9/32 patients (28%) after 35 cycles, including 4 patients who achieved CR, 2 PR, and 3 CI. In total, 12 TET2 mutations were identified in 9/32 patients (28%), of whom 5 had MDS/MPN (3 with CMML-1/2) and 4 had sAML. Unique compound heterozygosity was found in 3 patients; consequently biallelic inactivation of TET2 was found in 4 patients. For analyses, patients with partial and complete responses were compared with refractory patients and response was correlated with the presence of TET2 mutations. Among TET2-mutated patients, only 1 patient responded to therapy, whereas 8 additional patients, including 3 patients with biallelic inactivation of the TET2 gene, did not show any improvement (11%). In contrast, among patients with WT TET2 gene, responses were seen in 8/23 patients (35%; p=.19). While the cohort of treated patients was small, our preliminary results indicate that the presence of TET2 mutations may represent a negative predictor of response to demethylating agents. Disclosures No relevant conflicts of interest to declare.

The association between immune-mediated aplastic anemia (AA) and paroxysmal nocturnal hemoglobinuria (PNH) has been well documented, yet the related immune pathophysiology remains ill-defined. Response of AA patients with PNH clones to immunosuppressive agents such as anti-thymocyte globulin (ATG) and cyclosporine A provides clinical evidence for the involvement of the immune system in the evolution of PNH. The similar immunobiology of PNH and AA is also exemplified by the overrepresentation of HLA-DR*15 in AA, AA/PNH and PNH. To discern the relationship between T cell responses and effector mechanisms we applied a battery of immune tests to patients with these rare diseases. First, using T cell receptor Vβ flow cytometry in a cohort of patients with AA (N=42), AA/PNH (N=15), or PNH (N=18), we identified cytotoxic CD8 T cell (CTL) clonal expansions in 28/42 (66.7%), 9/15 (60%), and 7/18 (38.8%) patients, respectively. CD4 T cell expansions were present in 6/42 of AA (14.3%), 2/15 (13.3%) of AA/PNH, and 2/18 (11.1%) of PNH patients. We then studied whether expanded clones were associated with production of inflammatory cytokines; across the entire cohort, patients with clonal CD8 Vβ expansions demonstrated a significantly increased proportion of IFN-γ producing T cells as well as elevated levels of circulating Fas-L when compared to patients without clonal skewing (p=.032 CD4+IFN-γ+, p=.008 CD8+IFN-γ+, p=.097 sFAS-L). Even more pronounced was the increase in the proportion of IFN-γ producing CD4 T cells in patients with clonal CD4 Vβ expansions (p=.010). Furthermore, while a strong trend toward increased sFAS-L as detected by ELISA was found in patients with CD8 Vβ skewing vs. those without, patients with pronounced CD4 expansions did not produce elevated Fas-L levels, consistent with different effector mechanisms employed by CD4 vs. CD8 T cells. Based on these results we hypothesized that the presence of PNH clones will be associated with activation of immune effector mechanisms. Linear regression analysis of the size of the PNH clone vs. proportion of IFN-γ CTLs displayed a positive correlation that nearly reached statistical significance at α=0.05 (p=.067). A high proportion of CD4 IFN-γ cells (defined by a value above 95% mean confidence intervals of controls) was also associated with the presence of PNH (p=.048). Genetic analysis revealed further clues as to the increased propensity of patients with AA and PNH clones to produce elevated levels of IFN-γ; the hypersecretor genotype T/T for IFN-γ was over-represented in AA (28% vs. 10% in controls, p=.02) and correlated with presence of a PNH clone (35% vs. 14%, p=.01). An essential role of T cells in generating permissive conditions for the evolution of PNH clones is also supported by the immunogenetic relationship of PNH to HLA-DR*15, a relationship which was confirmed in our population of patients: phenotype frequency of HLA DR*15 was 42.8% AA, 40% AA/PNH, 27.8% PNH vs. 17.2% in control group. When HLA DR*15 positive and DR15 negative patients were compared, those with DR*15 displayed a strong trend toward increased proportion of CD8+IFN-γ producing cells (p=.094), previously shown to be elevated in patients with PNH clones. Our results reveal insights into the nature of permissive conditions involving oligoclonal T cell responses, oversecretion of proinflammatory cytokines, and immunogenetic background which together may promote the expansion of PNH clones. Conversely, it remains possible that the cytotoxic milieu may be a result of an immune response directed against intrinsically abnormal PNH clones.