Morice, William G.

Abstract Background: CD123 is the α-subunit of the high-affinity receptor for interleukin-3; its expression in normal bone marrow (BM) is highly restricted, however CD123 is highly expressed in certain hematolymphoid malignancies. In a pilot study of 11 systemic mastocytosis (SM) patients (Lasho et al., Leukemia, 2015), we made the following novel observations (i) CD123 was aberrantly expressed on neoplastic mast cells (MC); and (ii) there was focal proliferation of plasmacytoid dendritic cells (PDC) around MC aggregates, suggesting a tumor promoting role for PDC. Objectives: To study MC CD123 expression by IHC in a larger cohort of SM patients to ascertain: (i) whether there is a significant difference in CD123 expression between SM subtypes; (ii) whether focal PDC proliferation around MC aggregates is correlated with CD123 expression; and (iii) whether CD123 expression has prognostic value for overall survival (OS). Methods: The current study was approved by the Mayo Clinic institutional review board. SM was classified as per standard criteria. Archived BM slides were stained for tryptase and CD123 as previously described (Lasho et al., Leukemia, 2015). Slides were reviewed independently by 2 hematopathologists who were blinded with regards to SM subgroups and scored on the following pre-defined parameters: (i) CD123 expression yes/no; (ii) CD123 expression strong/weak; (iii) focal PDC proliferation yes/no; and (iv) BM MC%. Results: We studied 58 SM patients and 3 controls. Of the former, 23 had indolent SM (ISM), 10 aggressive SM (ASM), 23 SM with associated hematological disease (SM-AHD), and 2 had mast cell leukemia (MCL). Of the SM-AHD patients, 10 (43%) had CMML, 5 (22%) myeloproliferative neoplasm-unclassified (MPN-u), 3 (13%) PV, 2 (9%) acute myeloid leukemia, and 1 each had lymphoma, myeloma and myelodysplastic syndrome. The median follow up from diagnosis was 32 months (range 0.1 to 162). Over this period, 3 patients (5%) had leukemic transformation and 30 patients (52%) died. Concordance rates between hematopathologists for CD123 positive/negative, CD123 strong/weak, focal PDC proliferation yes/no and MC% were 97%, 94%, 98% and 100%, respectively. MC CD123 expression was absent in control cases but demonstrable in 37 (64%) SM patients; expression rates were 100%, 61%, 57%, and 0% in ASM, ISM, SM-AHD and MCL, respectively (p=0.02). Similar to MCL, both cases with SM-AML showed absent MC CD123 expression. Strong MC CD123 expression (comparable to background interstitial PDC) was observed in 23 patients (62%), including 85% with SM-AHD, 60% with ASM, and 43% with ISM (p=0.08). Focal PDC proliferation around MC aggregates, regardless of CD123 expression status, was noted in 44 patients (76%), including 90% with ASM, 87% with ISM, 65% with SM-AHD and 0% with MCL (p=0.02). Focal PDC proliferation was significantly higher in CD123-positive versus -negative patients (87% versus 50%, p=0.005). OS was predicted by SM subtype; the median OS for ISM, ASM, SM-AHD and MCL patients was not reached, 30 months, 25 months and 3 months, respectively (p=0.005). CD123 expression status did not have prognostic value for OS overall, however, in SM-AHD patients, CD123 expression was associated with significantly inferior survival (p=0.04), independent of the associated disease (ie, CMML vs AML vs MPN vs other). Survival outcome of SM-CMML patients expressing MC CD123 (n=3, median OS=11 months) was significantly inferior as compared to patients who did not (n=7, median OS=44 months), independent of age and absolute monocyte count (p=0.01). CD123 staining intensity had borderline significance for OS in the overall cohort (p=0.08). When restricted to non-indolent SM cases, strong CD123 expression was associated with significantly worse overall survival (15 versus 88 months, p=0.04). Conclusions: In this cohort, aberrant MC CD123 expression was observed in the majority of SM patients with highest prevalence in ASM patients and uniformly absent expression in leukemic phase SM (MCL or SM-AML). Focal PDC proliferation around MC aggregates was significantly higher in CD123-positive cases, indicating a potential functional interaction. CD123 expression identified SM-AHD patients with inferior OS, especially those with SM-CMML. Strong CD123 staining intensity may be associated with inferior OS in non-indolent SM patients. CD123 expression on MC and PDC constitutes a novel therapeutic target in non-leukemic SM. Disclosures Pardanani: Stemline: Research Funding. Brooks:Stemline Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties.

Abstract Background Prior studies have shown that presence of increased circulating plasma cell (PCs) identified using a slide-based immunofluorescence method is an adverse prognostic marker for overall survival in multiple myeloma (MM), and increases the risk of progression in patients with MGUS and smoldering MM. However, utility in clinical settings has been limited by the cumbersome nature of the test and lack of widespread availability. We studied the prognostic value of circulating PCs using sensitive multiparametric flow cytometry that enable us to quantitatively assess circulating PCs in patients with MM. Methods We evaluated all newly diagnosed MM patients seen at the Mayo Clinic, Rochester from 2009 to 2011 who had their peripheral blood samples evaluated by flow cytometry prior to therapy. Patients with plasma cell leukemia were excluded. Each blood sample had its peripheral blood mononuclear cells isolated by ficoll gradient and stained with antibodies to CD45, CD19, CD38, CD138 and cytoplasmic Kappa and Lambda Ig light chains. A six-color multi-parameter flow cytometer (Becton Dickinson FacsCantos II) was used to examine each sample with a target of detecting 150,000 events (cells) that was then analyzed using the Facs Diva Software. PCs were selectively analyzed through combinatorial gating using light scatter properties and CD38, CD138, CD19, and CD45. Normal PC's were then separated from clonal PCs based on the differential expression of CD45, CD19 and polytypic Ig light chains. The clonal PCs detected were reported out as the number of clonal events/150,000 collected total events. For those samples where less than 150,000 events were gated or examined, the number of final clonal events was adjusted to 150,000 events. Survival analysis was performed by the Kaplan-Meier method and differences assessed using the log rank test. Results There were 158 consecutive patients with newly diagnosed MM who had their peripheral blood evaluated via flow cytometry as part of their routine clinical evaluation. The median age of this group was 66 years (39-95) and 59% were male. At the time of this analysis, 25 patients had died and the 2-year OS rate for the cohort was 83%. The 2-year OS for the 89 (55%) patients with any circulating PCs was 76% compared with 91% for those with none (P=0.02). The median number of circulating clonal PCs was 33 (range, 0-46,413)/ 150,000 gated events. Using a ROC analysis the best cutoff predicting 1 and 2-year mortality were 435 and 376 events, respectively. Based on this, we defined >=400 events as a cutoff for defining patients with high-risk disease. The median time-to-next-treatment (TTNT) for patients with circulating clonal PCs >=400 (n=37, 23%) was 14 months compared with 26 months for those with <400 events (n=121, 77%) (p<0.001; Fig 1a). The median OS for those with >=400 clonal PC events was 32 months compared with not reached for those with <400 events (p<0.001; Fig 1b). Patients with >= 400 circulating PCs had higher ISS stage, creatinine, LDH, PC labeling index and bone marrow PC% compared with the rest. In a univariate analysis examining the presence of circulating PC >=400, LDH, labeling index, marrow PC% and FISH risk status, only circulating PCs were prognostic for overall survival (P = 0.0011). Among the 89 patients with any circulating PCs, 26 (30%) had CD45+ clonal PCs. The TTNT in patients with a clonal CD45+ population was inferior to those with no CD45+ clonal PCs in circulation (median 11 vs. 19 months, P = 0.034) as was the OS (P = 0.039). Conclusion Quantitative estimation of circulating clonal PCs in patients with newly diagnosed MM is a powerful predictor of early relapse from therapy and mortality. A cutoff of >=400 clonal events/150,000 gated mononuclear events predicts for a median TTNT of 14 months and OS of 32 months. This parameter is more powerful than the current high-risk parameters such as FISH risk status as well as traditional high-risk markers such as ISS stage and LDH levels, and is able to identify a group of patients with particularly poor outcome. Disclosures: Kumar: Celgene: Consultancy, Research Funding; Millennium: Consultancy, Research Funding; Onyx: Consultancy, Research Funding.

Abstract Abstract 2803 Poster Board II-779 Background. The rate of asymptomatic amyloidosis (asym-amyloidosis) detected in patients with newly diagnosed multiple myeloma (MM) or smoldering multiple myeloma (SMM) is unknown. This topic is significant because unrecognized AL may be associated with increased mortality may change the patient's management. The objective of the present investigation was to evaluate the number and clinical significance of asym-amyloidosis in MM and SM patients at the time of the diagnostic bone marrow (BM) biopsy for MM. Materials and Methods. The study population was selected from the Mayo Clinic Dysproteinemia database and consisted of consecutive patients with an established diagnosis of MM or SMM without recognition of symptomatic AL. Bone marrow biopsies at diagnosis of MM or SMM were retrospectively stained with Congo Red and reviewed by a single pathologist. A patient was considered to have asym-amyloid if Congo Red staining with apple green birefringence was found. Results. Biopsies from 144 (M 84, F 59) patients were evaluated: 77 had a diagnosis of MM and 67 of SMM. The median age was 59 (range 26-84) years. No differences were found regarding hemoglobin, platelets, prothrombin time, serum and urine proteins, serum albumin, alkaline phosphate, creatinine and β2-microglobulin among MM and SMM patients. At a median follow-up 76 months (range 0-216), 32% patients were alive, 65% dead and 2.7% lost to follow-up. Immunoglobulin isotypes were as follows: 96/144 (67%) had IgG 23/144 (16%) IgA, 12/144 (8%) had light chain only, 1/77 (1%) had IgD, none had IgM and 12/144 (8%) had biclonal or indeterminate; 84/144 (58%) were κ restricted. The presence of amyloid was found in only 2 cases (1%, 95% CI – 0.6 to 3.2), 1 in MM and 1 in SMM group. Neither of these patients had or developed signs or symptoms suggestive of organ involvement by amyloid. Among the 142 other patients without amyloid deposition in their index bone marrow, 1 (0.7%, 95% CI -0.6 to 2.0) developed symptomatic AL after 119 months of follow-up. Characteristics of these three patients are shown in table 1. Conclusions. We found only 2 cases (1%) of amyloidosis in the 144 cases of MM or SMM. Our estimates are lower than the rates which have been reported by others, perhaps because of our high level of suspicion for amyloid at our Amyloidosis Center. These data do not support the need for searching for asym-amyloidosis in patients with newly diagnosed MM or SMM as long as they have no clinical features of AL. Disclosures: Off Label Use: Hydroxyurea use in myelofibrosis. Gertz:celgene: Honoraria; millenium: Honoraria, Membership on an entity's Board of Directors or advisory committees. Witzig:Novartis: Research Funding. Kumar:celgene: Honoraria; millenium: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Abstract Background: The prognosis of patients with T-cell large granular lymphocyte (LGL) leukemia is diverse, with some patients experiencing rapid progression of the disease while others surviving decades. Despite multiple case reports and a number of case series reported in literature, prognostic factors in this leukemia are unknown and no staging/prognostic system has been developed. Aim: To identify factors predicting the prognosis of patients with newly diagnosed T-cell LGL leukemia. Methods: We reviewed the presentation, laboratory data and the clinical course of patients with newly diagnosed T-cell LGL at seen at the Mayo Clinic, Rochester. T-cell LGL was diagnosed in patients with at least 2 of these criteria: morphological evidence of LGL leukemia on bone marrow biopsy or blood smear; LGL immunophenotype; evidence of clonal T-cell receptor gene rearrangements. Results: 286 patients seen at Mayo Clinic Rochester between 6/1986 and 5/2005 met diagnostic criteria (52% male, median age at presentation 64 years (range 27–88)). Lymphadenopathy, splenomegaly, and hepatomegaly were present in 9%, 4% and 1.5% of the patients respectively. Median hemoglobin was 11.7 g/dL (range 3.5 – 17.1). Median absolute neutrophil count (ANC) was 1.51 × 109/L (range 0.0 – 20 × 109/L) and median absolute lymphocyte count (ALC) was 2.2 × 109/L (range 0.1 – 24). Median LGL count on peripheral smear was 0.81 × 109/L (range 0.06–10.8 × 109/L). Cytogenetic studies were done for 122 patients, of which, only 9 were abnormal. 87 patients (29%) had associated autoimmune conditions: rheumatoid arthritis (n=29), Felty’s syndrome (n=11), other connective tissue disorders (n=9)). Pure red cell aplasia, aplastic anemia, idiopathic thrombocytopenic purpura and autoimmune hemolytic anemia were seen in 17, 5, 8, 1 patients respectively. Median follow up was 50 months (range 1–229) and estimated median survival was 74.3 months. In multivariate analysis, the only independent risk factors were: anemia (hemoglobin<12 g/dl), hazard ratio (HR) 1.75, (95%CI 1.22–2.52, p= 0.0023), severe neutropenia (<0.1 × 109/L), HR 2.11 (95%CI 1.053–4.25) and lymphopenia (ALC< 1 × 109/L), HR 1.93 (95%CI 1.21–3.07). We developed a simple prognostic scoring system based on the above identified independent risk factors, stratifying patients in 3 groups: 0 - no risk factors present, 1 -one risk factor, 2 - two or more risk factors present. The median survival was 105, 55 and 5 months for score of 0, 1 and 2 respectively, p=0.004 (Figure). Conclusion: The prognosis of patients with newly diagnosed T-cell LGL is variable. The presence of anemia, severe neutropenia and lymphopenia are independent poor prognostic factors in T-cell LGL. Combining these factors allows the identification of patients at a high risk of death from the disease. Further studies are needed to prospectively validate these findings and establish if the high risk group would benefit from aggressive therapy. Figure Figure.

Abstract The peripheral blood absolute lymphocyte count (ALC) on day 15 after autologous stem cell transplantation (ASCT) has been shown to be an independent predictor for overall survival (OS) for many malignancies including acute myelogenous leukemia (AML), breast cancer, multiple myeloma (MM), primary systemic amyloidosis, and Hodgkin’s and non-Hodgkin’s lymphoma (NHL). However, due to the retrospective nature of previous studies, the peripheral blood lymphocyte subpopulations that predict survival are unknown. To prospectively correlate peripheral blood lymphocyte subpopulations after ASCT and progression free survival, peripheral blood lymphocytes collected from patients before and on day 15 after ASCT were analyzed by four color flow cytometry for CD3, CD4, CD8, CD16, CD 19, and CD56. Patients were then dichotomized into two groups: patients achieving normal numbers of lymphocyte subset (i.e. CD4, CD8, CD19, CD16/56) count versus those who did not. Progression free survival was then analyzed by the Kaplan-Mier method. In our cohort of 14 patients, 9 were male and 5 were female. Nine patients were diagnosed with diffuse large cell B cell lymphoma, two with mantle cell, two with follicular, and one with peripheral T cell lymphoma. On presentation, one patient had stage I, four patients had stage II, four had stage III, and five patients had stage IV disease. The median age at ASCT was 53 years (range: 26–70). The preliminary data from this ongoing prospective study of 14 patients shows that on day 15 after ASCT, 4/14 (29%) of patients achieved a normal CD3 count (median 321, range: 69–2069 cells/ml) 3/14 (21%) achieved a normal CD4 count (median 206, range: 31–1091 cells/ml), 6/14 (43%) achieved a normal CD 8 count (median 88.5, range: 13–813 cells/ml) 1/14 (7%) achieved a normal CD19 count (median 2, range:0–227 cells/ml), and 8/14 (57%) achieved a normal CD16/56 count (median 85.5, range: 10–744 cells/ml). The median follow-up was 12 months (range: 3–45 months). On univariate analysis, patients achieving an absolute NK cell count of ≥ 80 cells/μ on day 15 had significantly improved progression free survival compared to those who did not (not reached vs 3 months, p<0.006, respectively). Similar analysis evaluating the absolute CD3, CD4, CD8, and CD 19 count was not significant (p=0.1, p=0.258, p= 0.06, and p=0.55, respectively). To our knowledge, this is the first report detailing the critical role of NK cell immune reconstitution after ASCT in progression free survival. Figure Figure

Abstract Plasma cell (PC) proliferative disorders vary in behavior ranging from MGUS, a usually indolent disorder, to multiple myeloma, an aggressive disease with poor long-term survival. Detecting PC immunoglobulin (Ig) light chain restriction is commonly used to demonstrate PC clonality and establish the diagnosis. Furthermore, greater degrees of peripheral blood (PB) and/or bone marrow (BM) involvement are associated with more aggressive disease types and, therefore, are adverse prognostic indicators. Flow cytometric immunophenotyping (FCIP) is often used for detecting PC Ig light chain restriction. Shortcomings of FCIP as traditionally performed include its relative insensitivity and its underestimation of PC number. For these reasons, the FCIP PC clonality assessment in our laboratory is currently supplemented with a slide-based immunofluorescence (IF) technique. Recent advances in flow cytometry have led to the development of powerful instruments and reagents, potentially enhancing the ability of FCIP to analyze PC clones. In this study a newly available single tube, 6-color FCIP approach for detecting and enumerating monotypic PCs was compared to the current combined FCIP/IF assay. 94 PB and 124 BM samples were analyzed by combined FCIP/IF and single tube 6-color FCIP assays. For PB, FCIP/IF used 2-color CD38-PE/ CD45-PerCP FCIP to enumerate PCs and slide based IF to detect Ig light chain restriction. For BM FCIP/IF used two 4-color FCIP tubes (Tube1: cytoplasmic (cy)-kappa Ig-FITC/CD20-PE/CD45-PerCP/CD38-APC; Tube 2: cy-lambda Ig-FITC/CD56-PE/CD45-PerCP/CD38 APC) and slide-based IF to confirm clonality. These FCIP assays were performed on a FACSCaliber. The single tube, 6-color FCIP assay used the following fluorochrome-antibody combination: cy-kappa Ig-FITC/cy-lambda Ig-PE/CD138-PerCP-Cy5.5/CD38-APC/CD19-PE-Cy7/CD45-APC-Cy7 with data acquired on a FACSCanto. The results are summarized in Tables 1 & 2, the concordance between the methods was high. All false-negative 6-color FCIP results were attributable to the use of isotonic lysis for cell isolation. When limiting the comparison to the ficolled cells alsoused for FCIP/IF, 6-color FCIP was consistently more sensitive. The 6-color assay also allowed for the discriminition between CD19-positive polytypic PCs and CD19-negative monotypic PCs and the detection of CD45-positive monotypic PC subsets which were often present and which were not identified in the combined FCIP/IF approach. Estimates of PB disease burden generated from the 6-color technique were similar to those determined from the FCIP/IF data which have previously been shown to be of prognositic significance. 6-color FCIP allows for the rapid, sensitive detection of monotypic PCs. The ability of 6-color FCIP to detect phenotypically distinct clonal PC subsets enhances the sensitivity and may provide prognositically significant data through both more complete cell enumeration and charactarization. Given these advantages, 6-color FCIP may eventually supplant traditional FCIP methods of PC analysis. Method Comparsion in BM Bone Marrow 6-color FCIP 6-color FCIP Positive negative total Positive = clone detected; negative = no clone detected. FCIP/IF Positive 81(65%) 3(2%) 84 FCIP/IF Negative 5(4%) 35(28%) 40 Total 86 38 124 Method Comparsion in PB Peripheral Blood 6-color FCIP 6-color FCIP Positive negative total Positive = clone detected; negative = no clone detected. FCIP/IF positive 24(26%) 7(7%) 31 FCIP/IF negative 9(10%) 54(57%) 63 total 33 61 94

Abstract Background: Prior studies suggest that the presence of >5% polyclonal plasma cells (pPCs) among total plasma cells (PCs) within the bone marrow (BM) is associated with a longer progression-free survival, higher response rates, and lower frequency of high-risk cytogenetic abnormalities in patients with newly diagnosed multiple myeloma (MM). However, the incidence and prognostic utility of this factor in patients with relapsed and/or refractory MM has not been previously evaluated. Thus, we evaluated the prognostic value of quantifying the percentage of pPCs among the total PCs in the BM of patients with actively relapsing MM. Methods: We evaluated all MM patients with actively relapsing disease (biochemical and/or symptomatic) seen at the Mayo Clinic, Rochester, from 2012 to 2013, who had BM samples evaluated by seven-color multiparametric flow cytometry. All patients had at least 24 months of follow-up from the date of flow evaluation. Cell surface antigens were assessed by direct immunofluorescence antibodies for CD45, CD19, CD38, CD138, cytoplasmic Kappa and Lambda Ig light chains, and DAPI nuclear stain. The flow cytometry data was collected using the Becton Dickinson FACSCanto II instruments that analyzed 150,000 events (cells); this data was then analyzed by multi-parameter analysis using the BD FACS DIVA Software. PCs were selectively analyzed through combinatorial gating using light scatter properties and CD38, CD138, CD19, and CD45. Clonal PCs were separated from pPCs based on the differential expression of CD45, CD19, DAPI (in non-diploid cases), and immunoglobulin light chains. The percentage of pPCs was calculated in total PCs detected. Survival analysis was performed by the Kaplan-Meier method and differences were assessed using the log rank test. Results: There were 180 consecutive patients with actively relapsing MM who had BM biopsies analyzed via flow cytometry as part of their routine clinical evaluation. The median age of this group was 65 years (range: 40 - 87); 52% were male. At the time of this analysis, 104 patients had died, and the 2-year overall survival (OS) rate for the cohort was 58%. The median number of therapies received was 4 (range: 1 - 15). Of these patients, 61% received a prior ASCT, and almost all (99%) received prior regimens containing either immunomodulators or proteasome inhibitors. There were 55 (30%) patients with >5% pPCs among the total PCs in their BM. The median percentage of pPCs among total PCs in these 55 patients was 33% (range: 5 - 99). The median OS for those with >5% pPCs was not reached compared with 22 months for those with <5% pPCs (P = 0.028; Figure 1). Patients with <5% pPCs PCs had a higher likelihood of high-risk FISH cytogenetics compared with the rest of the patients. In a univariate analysis, increasing number of pPCs was associated with an improved OS, while higher labeling index, number of prior therapies, and the presence of high-risk FISH cytogenetics were associated with a worse OS. In a multivariate analysis, only the increasing number of pPCs (P = 0.006), higher labeling index (P = 0.0002) and number of prior therapies (P = 0.003) retained statistical significance. Conclusion: Quantitative estimation of the percentage of pPCs among the total PCs in the BM of patients with actively relapsing MM was determined to be a predictor of worse OS. As such, this parameter is able to identify a group of patients with MM with actively relapsing disease who have a particularly poor outcome. Further studies evaluating its biological significance are warranted. Figure 1 Kaplan-Meier curve comparing OS between patients with ≥5% pPCs and <5% pPCs among the total PCs in their BM. Figure 1. Kaplan-Meier curve comparing OS between patients with ≥5% pPCs and <5% pPCs among the total PCs in their BM. Disclosures Kapoor: Celgene: Research Funding; Amgen: Research Funding; Takeda: Research Funding. Gertz:Prothena Therapeutics: Research Funding; Novartis: Research Funding; Alnylam Pharmaceuticals: Research Funding; Research to Practice: Honoraria, Speakers Bureau; Med Learning Group: Honoraria, Speakers Bureau; Celgene: Honoraria; NCI Frederick: Honoraria; Sandoz Inc: Honoraria; GSK: Honoraria; Ionis: Research Funding; Annexon Biosciences: Research Funding. Kumar:AbbVie: Research Funding; Noxxon Pharma: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Array BioPharma: Consultancy, Research Funding; Sanofi: Consultancy, Research Funding; Onyx: Consultancy, Research Funding; Skyline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Research Funding; Kesios: Consultancy; Glycomimetics: Consultancy; BMS: Consultancy.

Abstract Peripheral T- and NK-cell lymphomas (PTCLs) are fatal in the majority of patients, and conventional chemotherapy is minimally effective. Targeted inhibition of tyrosine kinases (TKs) might be a useful treatment strategy, but TK overexpression by PTCLs has not been well characterized (except ALK). Recently a novel translocation t(5;9) fusing the ITK and SYK genes was reported in a subset of unspecified-type PTCLs, associated with high SYK gene expression and Syk protein positivity. Syk is a non-receptor TK important in immunoreceptor signal transduction, providing proliferation and survival signals in a variety of hematopoietic cells. Syk is absent or expressed at very low levels in most normal post-thymic T cells, where this signal transduction role is fulfilled by ZAP70. Since Syk TK inhibitors are now in clinical trials for other diseases, we sought to determine the incidence of t(5;9) and Syk overexpression in PTCL. Tissue microarrays were constructed using triplicate cores from paraffin-embedded tissue blocks. Cases were studied using fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). Additional studies were performed using flow cytometry and Western blot. There were 141 PTCLs, including 35 angioimmunoblastic, 66 unspecified, 21 anaplastic large cell, 2 enteropathy-associated, 5 hepatosplenic, 5 extranodal NK/T, and 7 other. No evidence of t(5;9) was identified by FISH in 86 informative cases. Normal T-cell subsets from peripheral blood, spleen, and lymph node were negative for Syk by IHC, flow cytometry, and Western blot. Syk was positive by immunohistochemistry in 133 of 141 (94%) PTCLs. All 8 Syk negative cases were extranodal, cytotoxic PTCLs. Western blot on frozen tissue correlated with IHC results in paraffin. All 4 Syk-positive cases tested showed phosphorylation of tyrosine residues 525/526, known to be required for Syk activation. We conclude that Syk is overexpressed in most PTCLs, including the vast majority of nodal cases. Syk may substitute for ZAP70 in these tumors and provide ongoing proliferation and survival signaling. The overexpression of Syk and phosphorylation of its Y525/526 residues suggest Syk may be a suitable target for pharmacologic TK inhibition as a treatment option for patients with PTCL.

Abstract Background: Proliferation rate of plasma cells (PC) is a powerful tool in evaluating all types of monoclonal gammopathies, predicting for progression as well as survival in all phases of the disease. A flow cytometry (FC)-based method to delineate the cell cycle of clonotypic PCs and determine the proportion in S-phase was developed. This method replaces a labor-intensive slide based methodology, but the prognostic power of this FC approach has not been clearly elucidated. Methods: A total of 1061 patients who had bone marrow (BM) FC PC proliferation analysis at Mayo Clinic, Rochester from May 2012 to December 2013 were studied. These included MGUS/ SMM (n=134), newly diagnosed (NMM; n=88), plateau in response (>=PR; n=544), and stable disease (n=38), and relapsed disease (RMM; n=257). PC proliferation was measured by 8-color FC using antibodies to CD19, CD38, CD138, CD45, and cytoplasmic kappa and lambda Ig light chains, and DAPI DNA staining. The DNA content of the abnormal, clonotypic PCs was specifically analyzed and the %S-phase was determined by measuring the proportion of cells between G0/G1 and G2/M. All studies collected 5x105 events on BD FACSCanto II instruments and analysis was performed with BD FACSDiva software. The CV of the DAPI staining of G0/G1 lymphocytes as an internal control population was always less than 3.5. Patients with fewer than 300 PCs for assessment of %SP were designated as 0%. Results: The median follow up from testing for the entire cohort was 13 months (95% CI; 12.7, 13.3); 156 (15%) had died. The proportion of patients with adequate numbers of plasma cells available for %SP estimation and the median (IQR) %SP for the different groups are as shown in table. As expected, patients with MGUS/SMM and those in a response state had slightly lower proportions of patients with sufficient PCs to estimate %SP. The clinical features and outcomes were further analyzed using two cutoffs, %SP ≥1 and ≥2, in the different patient groups. Among most of the disease groups, especially the newly diagnosed MM and relapsed disease, %SP was prognostic for OS using both cutoffs (Table). Among the NMM in univariate analysis, %SP ≥2, B2microglobulin >5.5, age >70 and creatinine > 2 mg/dl were all significant, but only B2microglobulin >5.5 retained significance in multivariable analysis. Among the RMM, in univariate analysis, %SP ≥2, B2microglobulin >5.5, HR FISH (t4; 14, t14;16, t14;20 & del17p) and LDH >221 were all significant, but only %SP ≥2 was significant in multivariable analysis. Conclusion: The %S-phase of clonal PCs determined by FC is of prognostic value in patients with MM, in all disease phases. This is particularly relevant among patients with relapsed disease, where it is independent of some of the other known poor prognostic factors such as high risk FISH. This is in sharp contrast with recent studies that have shown no prognostic value for slide based labeling index in patients treated with novel agents. Abstract 2053. Table MGUS/SMM NMM >=PR SD Relapse N 134 88 544 38 257 N (%) with PCs measurable for proliferation 103 (77%) 84 (96%) 245 (45%) 31 (82%) 210 (82%) %SP (IQR) 0.3(0.1-0.7) 0.8 (0.4- 1.5) 0(0- 0.9) 0.8(0.3-1.3) 1.2(0.4- 2.7) OS (%SP >1 vs. rest) NS NR for either(P<0.01) NR for either (P<0.001) NR for either(P=0.02) 19 vs. NR(P<0.001) OS (%SP >2 vs. rest) NS NR for either (P=0.03) 20 vs. NR(P<0.001) NR for either(P=NS) 16 vs. NR(P<0.001) Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.