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Abstract Background: Cutaneous T-cell lymphoma (CTCL) is an incurable lymphoproliferative disorder characterized by the clonal expansion of malignant CD4+ T cells. Currently there is a poor understanding of the underlying mechanisms in the development and progression of this disease. Members of the TNF superfamily have been shown to be important mediators in the formation and regulation of normal T cell responses. A proliferation-inducing ligand (APRIL), a newly identified member of the TNF superfamily, has been shown to stimulate tumor cell growth as well as proliferation of primary B and T lymphocytes. APRIL transcript levels are reported to be low in normal tissues with the highest levels found in peripheral blood leucocytes. In contrast, higher mRNA levels have been detected in tumor cell lines and primary tumor tissues and various tumor cell lines have been shown to respond to APRIL with increased tumor cell proliferation and decreased apoptosis. The striking effects of APRIL on tumor cell growth and survival raise the possibility that the APRIL and its receptors-TACI and BCMA may be involved in the pathogenesis and maintenance of T cell malignancies. Goal: To explore the involvement of APRIL on normal and malignant T cell growth and survival. Results: Under resting conditions, there was minimal APRIL binding to normal T-cells and no TACI or BCMA expression. However, stimulation of resting T cells with OKT3 (anti-CD3) or a combination of PMA and ionomycin resulted in an increase in APRIL binding and TACI expression on a subset of activated T-cells. When CD3+ resting human T cells were cultured in the presence of APRIL, IL-2, or OKT3 alone or a combination, normal T cells had greater viability compared to the nil control and APRIL mediated-T cell survival was similar level to that seen with IL-2 or OKT3 alone. CSFE labeled T cells were activated with suboptimal dose of OKT3 and incubated in the presence of APRIL and /or IL-2. Both APRIL and IL-2 alone induced cell proliferation, while treatment with the combination of APRIL and IL-2 had a synergistic effect on T cell proliferation. Malignant T cells isolated from the blood of patients with Sezary Syndrome exhibited a similar pattern of APRIL binding and APRIL also enhanced their survival. Gene array experiments were performed to identify genes involved in APRIL-mediated survival. In normal and primary malignant T cells the anti-apoptotic genes BCL2A1, BIRC6, CHK1, MCL-1, and MDM2 were upregulated and the pro-apoptotic genes CASP5, DR5, and TNFR2 were downregulated in response to APRIL. Conclusion: APRIL promotes the survival of normal and malignant T cells and co-stimulates CD3-mediated T cell proliferation. The gene array experiments suggest that APRIL regulates the expression of anti-apoptotic and pro-apoptotic genes in both normal and malignant T cells. Taken together, these data indicate an important role for APRIL in the biology of normal T cells and in the pathophysiology of T cell lymphoma.

Abstract Background: Patients with newly diagnosed, advanced-stage, follicular B-cell non-Hodgkin’s lymphomas (NHL) commonly receive rituximab as initial therapy. While antibody dependent cytotoxicity (ADCC) is felt to be the predominant mechanism of action for rituximab, the immunologic effects of rituximab are largely unknown. We have previously shown that genetic polymorphisms in IL-8, IL-2, IL-12 and IL-1 receptor antagonist (IL-1RN) have prognostic significance in follicular lymphoma. The goal of this study was to measure changes in the abovementioned and other serum cytokine levels in patients receiving single agent rituximab as initial therapy for follicular NHL and to identify cytokines that are associated with an increased risk of disease relapse. Methods: Patients with untreated follicular grade 1 NHL, and measurable stage III/IV disease were treated with rituximab 375 mg/m2 intravenous weekly for 4 doses and were then followed for response and time to progression (TTP); no maintenance therapy was provided. Thirty-seven patients were enrolled in this clinical trial between July 1999 and May 2001. As previously reported, the overall response rate in the 36 eligible patients was 72%, with 36% complete remissions. Now, with a median follow up of 5.1 years, the median TTP is 2.2 years (95% CI, 1.2–3.3 years) and the 5-year overall survival is 77% (95% CI: 64–92%). We measured the serum levels of a variety of inflammatory cytokines by ELISA at baseline and 1 month after completing treatment. Serum specimens were available in 30 patients. Thirty two cytokines or receptors including IL-1β, IL-1RN, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 p40/p70, IL-13, IL-15, IL-17, TNF-α, IFN-α, IFN-γ, GM-CSF, MIP-1α, MIP-1β, IP-10, MIG, Eotaxin, RANTES, MCP-1, VEGF, GCSF, EGF, FGF-basic, HGF, BLyS and APRIL were analyzed. Results: A significant increase was seen in the serum levels of BLyS (p=0.0001) after rituximab therapy, while IL-12 (p=0.0002), IL-1RN (p=0.0001), IL-2R (p=0.0001), IL-1β (p=0.02), IL-7 (p=0.02), IL-10 (p=0.02), and MIG (p=0.05) all significantly decreased. All other cytokines/receptors remained unchanged. Higher pretreatment serum levels of IL-2R (p=0.008) and IL-12 (p=0.001) correlated with a shorter TTP. Furthermore, elevated serum IL-2R levels pretreatment correlated with a lack of response to rituximab (p=0.03). Differences in overall survival could not be determined due to the fact that only 8 patients enrolled in the study have died. Because serum IL-2R levels correlated with response and TTP, we measured the cell surface expression of IL-2R in the tumor by flow cytometry. IL-2R was detected on a subpopulation of CD4+ T-cells in all specimens tested (n=14), and on malignant B-cells in 5 cases (36%). Conclusions: Pretreatment serum levels of IL-2R and IL-12 are important prognostic factors for response to therapy or TTP in follicular lymphoma patients treated with rituximab. Furthermore, serum levels of BLyS, IL-1RN, IL-7, IL-10, IL-1β and MIG also change after rituximab treatment. Targeting these cytokines may therefore provide additional clinical benefit to patients treated with rituximab.

Recent studies have shown decreased megakaryocyte expression of the thrombopoietin receptor (c-mpl) in patients with polycythemia vera (PV) but not in those with reactive erythrocytosis. We examined the diagnostic utility of this observation in 22 patients with PV, 7 patients with secondary erythrocytosis (SE), and 10 normal controls. Commercial antibodies against c-mpl were used with standard immunoperoxidase methods. Megakaryocyte c-mpl staining intensity was uniformly moderate-to-strong in the healthy controls and in all the patients with SE. In contrast, staining intensity in 9 patients with PV (41%) was uniformly weak. Furthermore, in 12 of the remaining 13 patients with PV, the c-mpl staining pattern in each case was heterogeneous and was associated with weak staining intensity in more than 20% of the megakaryocyte population. These preliminary data suggest that c-mpl immunostains may complement bone marrow histopathology in distinguishing PV from nonclonal causes of erythrocytosis.

Abstract Background: Almost all cases of polycythemia vera (PV) and more than half of those with either essential thrombocythemia (ET) or primary myelofibrosis (PMF) harbor JAK2V617F. It has been previously shown that V617F-positive ET patients, as opposed to their mutation-negative counterparts, require lower doses of hydroxyurea (HU) for control of their platelet count. In the current study we looked for a similar scenario in PMF and PV. Methods: Study patients were identified by database inquiry based on the presence of treatment history with HU and adequate follow-up. Qualitative (PMF) and quantitative (PV) analysis of JAK2V617F was performed according to previously published allele specific PCR-based methods. Mutation analysis was performed on bone marrow samples collected at time of initial diagnosis or within one year of diagnosis. HU treatment response assignment was according to criteria set by the International Working Group for Myelofibrosis Research and Treatment. In PV, a complete response (CR) required normalization of blood counts associated with a more than 50% benefit in both phlebotomy requirement and splenomegaly. Partial response (PR) required either becoming phlebotomy independent or normalization of blood counts or a greater than 50% improvement in splenomegaly. Results: The study cohort consisted of 69 patients with PMF (median age 62 years; 36% females) and 56 patients with PV (median age 67 years; 45% females). In PMF, by univariate analysis, HU response was associated with a shorter disease duration (p=0.008), absence of previous therapy (p=0.01), older age at diagnosis (p=0.009), and presence of V617F (p=0.02). On multivariable analysis, only the latter retained its significance; HU response rate was 48% in V617F-positive patients (11/21 patients) and 8% in mutation-negative cases (1/12 patients). In PV, V617F allele burden correlated directly with HU response (p=0.05) and, in responding patients, inversely with HU dose (p=0.01; Figure). All 9 patients with upper quartile V617F allele burden responded (8 with CR) whereas 6 of the 11 (55%) HU non-responders displayed V617F allele burden in the lower quartile range. Among the 45 HU responders, the maximum daily dose of HU employed in all 9 patients in the upper quartile allele burden range and 4 of the 5 in the middle quartiles range was below 1500 mg. In contrast, 8 of the 31 patients (26%) in the lower allele burden quartile range required doses at 1.5 gm or more per day. Conclusion: The current study suggests that response to HU therapy in both PMF and PV is more robust in the presence of JAK2V617F; the particular information is useful in identifying PMF patients who are likely to benefit from HU therapy and in individualizing HU starting dose in PV patients. Figure Figure

Abstract Abstract 3903 Poster Board III-839 Background A peripheral blood (PB) absolute eosinophil count (AEC) of ≥ 1.5 × 109/L is operationally referred to as “hypereosinophilia”. In general, one can classify hypereosinophilia into 3 major categories: i) secondary, ii) clonal, and iii) idiopathic. The 2008 WHO proposal provides a modern-era framework for the classification of clonal eosinophilia and subcategories include (i) myeloid/lymphoid neoplasms associated with eosinophilia and PDGFRA-, PDGFRB-, or FGFR1-rearrangement; (ii) clonal eosinophilia associated with an otherwise WHO-defined myeloid malignancy such as systemic mastocytosis (MN-eo), and (iii) chronic eosinophilic leukemia, otherwise not specified (CEL-NOS). Idiopathic hypereosinophilia (IH) with organ damage and without clonal/abnormal lymphocytes is referred to as hypereosinophilic syndrome (HES). In the current study, we define the spectrum of disease phenotypes and survival in 357 consecutive patients with hypereosinophilia seen at a single institution. Methods Mayo Clinic patient records from January 1994 to October 2008 were reviewed, and those with persistent eosinophilia ≥1.5 × 109/L identified. Clinical data and bone marrow (BM) histology were reviewed, and the eosinophilic disorder classified as above. The aims of the current study were to: (i) study the spectrum of eosinophilic disorders within the context of the WHO proposal; (ii) describe the clinical, laboratory, and molecular/cytogenetic characteristics of patients with primary eosinophilia (i.e. clonal and idiopathic); and (iii) assess the prognostic relevance of the WHO proposal from the standpoint of life expectancy and risk of leukemic transformation. Results (i) Disease spectrum and clinical characteristics: A total of 357 cases (median age=52 years) were identified: (i) secondary eosinophilia 165 (46%; male ∼52%) (autoimmune 33%, non-myeloid malignancy 19%, drugs 12%, allergies 10%, parasitosis 2%, others 24%); (ii) PDGFRA- or PDGFRB-rearranged myeloid neoplasm 17 (5%; male 100%); (iii) CEL-NOS 6 (2%; male 67%); (iv) MN/eo 36 (10%; male 69%) (systemic mastocytosis 50%, chronic myeloproliferative neoplasm 33%, AML 6%, others 11%); (v) IH 115 (32%; male 58%); and (vi) eosinophilia associated with clonal/abnormal lymphocytes 14 (4%; male 57%). Of the 115 patients with IH, 72 (63%) had evidence of end-organ damage, and could be classified as having HES: 36 patients had 1 organ involved, 21 had 2, and 15 had 3-4. In HES patients, the major organs involved were: lung 23%, skin/mucosa 22%, cardiac 14%, nervous system 10%, and thrombosis 9%. (ii) Molecular and cytogenetic abnormalities: Complete karyotype information was available for 144 (77%) patients; 21 (15%) patients had an abnormal karyotype; MN/eo 12 (35%), PDGFRA- or PDGFRB-rearranged myeloid neoplasm 4 (31%), CEL-NOS 5 (83%), IH and eosinophilia associated with clonal/abnormal lymphocytes 0 (p<0.01). Of the 126 (67%) patients screened for FIP1L1-PDGFRA, 16 (13%) tested positive. Two patients (1%) harbored t(5;12). Of the 108 (57%) patients screened for presence of an occult T-cell clone (PCR for TCR gene rearrangement), 14 (13%) tested positive. (iii) Survival and risk of disease transformation: At a median follow of 21 months (range <1-174), 56 (30%) deaths were recorded; MN/eo 23 (64%), IH 25 (22%), PDGFRA- or PDGFRB-rearranged myeloid neoplasm 3 (18%), CEL-NOS 3 (50%), and T-cell/eo 2 (14%). The combined median survival was 95 months: MN/eo 34 months, CEL-NOS 13 months, IH 151 months, PDGFRA- or PDGFRB-rearranged myeloid neoplasm and eosinophilia associated with an abnormal lymphocyte clone (not reached) (p<0.01; Figure). Transformation to acute leukemia was seen in 4 patients (2 with PDGFRA- or PDGFRB-rearranged myeloid neoplasm, and 1 each with CEL-NOS and IH) (p<0.01). Conclusions In a major tertiary center in the United States, the two major causes of hypereosinophilia were secondary (46%) and idiopathic (32%). Regarding the former, autoimmune diseases and parasitic infections were the most and least frequent causes, respectively. Cases with clonal eosinophilia were predominantly male while the sex ratio was more balanced in the other subcategories. Among primary hypereosinophilia, survival was the worst in clonal eosinophilia not associated with the imatinib-sensitive PDGFR mutations. Additional data regarding clinical characteristics, laboratory findings, and treatment outcome of idiopathic hypereosinophilia patients will be presented at the meeting. Disclosures: Pardanani: TargeGen: Research Funding; Cytopia: Research Funding.

This study evaluated the contributing roles of flow cytometric immunophenotyping of blood and bone marrow and immunohistochemical paraffin section staining of bone marrow biopsies in the staging of B-cell malignant lymphoma. Flow immunophenotyping was performed on a marrow specimen in 175 cases; a corresponding blood specimen was also immunophenotyped in 135 of these cases. Morphologic marrow involvement by lymphoma was found in 59 cases; flow immunophenotyping identified 54 cases with a monoclonal B-cell process: morphology-positive/flow-positive (n = 49), morphology-positive/flow-negative (n = 10), morphology-negative/flow-positive (n = 5), and morphology-negative/flow-negative (n = 111). The 10 morphology-positive/flow-negative cases included 5 follicular and 5 large-cell lymphomas with minimal marrow involvement. All 5 morphology-negative/flow-positive cases were from patients with large-cell lymphomas and bulky clinical disease. Because the blood contained the same B-cell clone in 2 of 2 morphology-negative/flow-positive cases studied, blood contamination of marrow may account for these findings. Blood flow cytometric immunophenotyping studies were positive in 32 cases; 30 had marrow involvement by morphology and were from patients with follicular, mantle cell, lymphoplasmacytic, small lymphocytic, or marginal zone lymphomas. From our results, we conclude that (1) bone marrow flow cytometric immunophenotyping is not a cost-effective replacement for good morphologic evaluation in lymphoma staging and that (2) a positive peripheral blood flow cytometric immunophenotyping study when performed in low-grade lymphomas correlates with marrow involvement.

Abstract Background: In both polycythemia vera (PV) and essential thrombocythemia (ET), aspirin and/or hydroxyurea therapy provide adequate protection from thrombosis but not from disease transformation into acute myeloid leukemia/myelodysplastic syndrome (AML/MDS) or myelofibrosis (MF). In designing clinical trials with new drugs, including the use of small molecule JAK2 inhibitors, in either ET or PV, it is important to identify patients who are at a higher risk of early transformation into AML/MDS/MF as well as those in whom the long-term risk of these complications is too low to justify exposure to experimental drugs with unknown short-term and long-term toxicity profile. Methods: Information on an institutional database of 1061 patients with either PV (n=458) or ET (n=603) was updated in July of 2007. The World Health Organization criteria were used for diagnosis of PV, ET, AML, MDS, and MF. Two different approaches were used to assess both early and overall risk of AML/MDS/MF development in both ET and PV. In the first approach, three distinct groups were delineated and their presenting features compared; group A was comprised of patients who have remained AML/MDS/MF free after a minimum follow-up of 20 years whereas groups B and C included patients who developed either AML/MDS or MF, respectively, in the first decade of their disease. In the second approach, prognostic factors for AML/MDS/MF-free survival were examined among the entire cohort of 1061 patients. Results : In ET, the number of patients that fulfilled the above-mentioned criteria for inclusion in groups A, B, and C were 40, 12, and 8; the three groups displayed significant differences in the prevalence, at diagnosis, of lower than normal hemoglobin level (8% vs. 58% vs. 38%; p=0.0004), male sex (18% vs. 58% vs. 50%; p=0.01), and arterial thrombosis at diagnosis (p=0.03), respectively. However, only the former two remained significant during multivariable analysis that included age as a covariate. In PV, the number of patients in groups A, B, and C were 23, 18, and 12; the only difference between the three groups was the association between group B and leukocytosis (p=0.02). Cox regression analysis of the entire 1061 ET/PV patients cohort, including 65 AML/MDS/MF events in ET and 36 AML/MDS events in PV, confirmed the inferior AML/MDS/MF-free survival in ET associated with lower than normal hemoglobin and AML/MDS-free survival in PV associated with leukocytosis (Figure). Conclusion : The current study identifies PV patients with leukocytosis as being the most appropriate and ET patients without anemia as the least appropriate for consideration of participation in experimental drug treatment trials. Figure Figure Figure Figure

Abstract Numerous clinical therapies have attempted to modulate tumor cell immunity, but for the most part, have proven unsuccessful. The inability to produce or augment an effective immune response is due in part to regulatory T (Treg) cells, which inhibit CD4 and CD8 T cell function. Our group has recently shown that Treg cell numbers are elevated in NHL tumors and that NHL B cells induce the development of Treg cells thereby inhibiting anti-tumor responses. The ability of NHL B cells to direct the cellular composition of their microenvironment is critical to our understanding of tumor immunity and we therefore wanted to determine if NHL B cells also directed the expansion or reduction of other T cell populations. IL-17-secreting CD4+ T cells (TH17), a newly characterized CD4+ T helper cell lineage, promote inflammation and play an important role in autoimmune disease. IL-17 has been shown to inhibit tumor cell growth suggesting a potential role for TH17 cells in anti-tumor immunity. We therefore set out to determine if TH17 cells were present in NHL tumors and whether or not their numbers were regulated by NHL B cells. Using unsorted mononuclear cells from malignant lymph nodes, we were unable to detect IL-17 expression in resting CD4+ T cells or CD4+ T cells activated with PMA/Ionomycin stimulation (less than 1%). However, IL-17-secreting CD4+ T cells could be detected in significant numbers in inflammatory tonsil and normal PBMCs. Interestingly, depletion of CD19+ NHL B cells from mononuclear cells obtained from patient biopsies resulted in detection of a clear population of IL-17-secreting CD4+ T cells (5%). These results suggest that NHL B cells suppress TH17 cell differentiation. The frequency of IL-17-secreting CD4+ T cells could not be further enhanced by the addition of exogenous TGF-b and IL-6, a cytokine combination favoring for TH17 differentiation, suggesting a further impairment of TH17 cell differentiation in the tumor microenvironment. In contrast, Foxp3 expression could be detected in resting CD4+ T cells (30%) and could be induced in CD4+CD25−Foxp3− T cells activated with TCR stimulation (28%). Contrary to the inhibition of TGF-b-mediated TH17 differentiation, Foxp3 expression could be dramatically upregulated by TGF-b in intratumoral CD4+ T cells (35%). In addition, lymphoma B cells strongly enhanced Foxp3 expression in intratumoral CD4+CD25−Foxp3−. Furthermore, when added together, the frequency of Foxp3+ T cells and Foxp3-inducible cells reached up to 60% of CD4+ T cells in tumor microenvironment of B-cell NHL. These findings suggest that the balance of effector TH17 cells and inhibitory Treg cells is disrupted in B-cell NHL and significantly favors the development of inhibitory Treg cells. Our data indicate that lymphoma B cells are key factor in regulating differentiation of intratumoral CD4+ T cells toward inhibitory CD4+ T cells.

Abstract Background The role of intratumoral PD-1+ T cells and their contribution to patient outcome in follicular lymphoma (FL) has been controversial. While some studies have found that increased numbers of PD-1+ T cells correlate with an inferior prognosis or have no impact on patient outcomes, other studies observed that the numbers of intratumoral PD-1+ T cells are an indicator of a favorable outcome in FL. In previous work, we observed that PD-1 can be expressed by different subsets of intratumoral T cells in FL including T follicular helper (TFH) cells and exhausted T effector cells. However, the function, prevalence, distribution and clinical importance of the respective intratumoral PD-1+ T subsets in FL patients are largely unknown. Goal To characterize PD-1+ T cell subsets and to determine the biological and clinical relevance of these T cell subsets in FL. Results In an analysis of biopsy specimens from 33 previously untreated FL patients, we found that PD-1 is abundantly expressed on intratumoral T cells. A median of 54.9% (range: 17.2-92.6, n=33) of CD4+ or 45.8% (range: 12.8-81.7, n=33) of CD8+ T cells express PD-1 on the cell surface. We found by flow cytometry that PD-1 is expressed on intratumoral CD4+ T cells with bright or dim intensity that represents two different subpopulations in FL. By immunohistochemistry, we identified that CD4+PD-1bright T cells predominantly reside in the lymph node follicles while PD-1dim T cells are mainly located in an inter-follicular pattern. Intratumoral CD4+PD-1bright T cells have a TFH cell phenotype and express CXCR5, secret IL-21 and are BCL-6 positive with no TIM-3 expression. In contrast, CD4+PD-1dim T cells have an exhausted phenotype, express TIM-3 and do not express BCL-6 and CXCR5. Functionally, CD4+PD-1dim T cells displayed reduced proliferation, cytokine production and cell signal transduction. When the CD8+ T cells were analyzed, we found that intratumoral CD8+ T cells, primarily reside in the inter-follicular regions, express PD-1 with low intensity and exhibit similar phenotypic and functional changes to inter-follicular CD4+PD-1dim T cells. These cells express TIM-3 but lack CXCR5 and BCL-6. They also displayed reduced proliferation and cytokine production. Clinically, we observed that both the numbers of CD4+PD-1dim and CD8+PD-1dim exhausted T cells significantly correlate with a reduced progression-free survival in FL patients treated with single agent rituximab (p=0.007 and p=0.04 respectively; n=31). Although it did not reach statistical significance, CD4+PD-1bright TFH cells appeared to correlate with a favorable survival, suggesting that TFH and exhausted T cells differentially impact patient outcome in FL. Conclusion Taken together, these results indicated that PD-1 expression defines two subpopulations with distinct functions that differentially impact patient outcome in FL. This finding provides not only an explanation for the previously discrepant clinical observations, but is also important in the design of future anti-PD-1 antibody-based therapy in FL. Disclosures: No relevant conflicts of interest to declare.

Abstract Recent studies have shown decreased megakaryocyte expression of the thrombopoietin receptor (c-mpl) in patients with polycythemia vera (PV) but not in those with reactive erythrocytosis. We examined the diagnostic utility of this observation in 22 patients with PV, 7 patients with secondary erythrocytosis (SE), and 10 normal controls. Commercial antibodies against c-mpl were used with standard immunoperoxidase methods. Megakaryocyte c-mpl staining intensity was uniformly moderate-to-strong in the healthy controls and in all the patients with SE. In contrast, staining intensity in 9 patients with PV (41%) was uniformly weak. Furthermore, in 12 of the remaining 13 patients with PV, the c-mpl staining pattern in each case was heterogeneous and was associated with weak staining intensity in more than 20% of the megakaryocyte population. These preliminary data suggest that c-mpl immunostains may complement bone marrow histopathology in distinguishing PV from nonclonal causes of erythrocytosis.