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Myeloid/lymphoid neoplasm with eosinophilia (MLN-Eo) is a World Health Organization (WHO) established category of hematologic malignancies primarily arising in adults. We discuss an 8-month-old infant who presented with clinical features similar to those of juvenile myelomonocytic leukemia (JMML) but who was diagnosed with MLN-Eo driven by an ETV6-FLT3 fusion. Results of patient-derived leukemia ex vivo studies demonstrated increased sensitivity to type I FLT3 inhibitors as compared with type II inhibitors. Treatment with the type I inhibitor gilteritinib resulted in complete immunophenotypic and cytogenetic remission. This patient subsequently underwent a hematopoietic stem cell transplant and remains in complete remission 1 year later. This is the youngest patient reported with an ETV6-FLT3 fusion and adds to the mounting reports of FLT3-rearranged MLN-Eo, supporting its addition to the WHO classification. Furthermore, this case highlights the clinical utility of ex vivo drug testing of targeted therapies.

Plasmacytoid dendritic cells (pDCs) are the principal natural type I interferon–producing dendritic cells. Neoplastic expansion of pDCs and pDC precursors leads to blastic plasmacytoid dendritic cell neoplasm (BPDCN), and clonal expansion of mature pDCs has been described in chronic myelomonocytic leukemia. The role of pDC expansion in acute myeloid leukemia (AML) is poorly studied. Here, we characterize patients with AML with pDC expansion (pDC-AML), which we observe in ∼5% of AML cases. pDC-AMLs often possess cross-lineage antigen expression and have adverse risk stratification with poor outcome. RUNX1 mutations are the most common somatic alterations in pDC-AML (>70%) and are much more common than in AML without pDC expansion and BPDCN. We demonstrate that pDCs are clonally related to, as well as originate from, leukemic blasts in pDC-AML. We further demonstrate that leukemic blasts from RUNX1-mutated AML upregulate a pDC transcriptional program, poising the cells toward pDC differentiation and expansion. Finally, tagraxofusp, a targeted therapy directed to CD123, reduces leukemic burden and eliminates pDCs in a patient-derived xenograft model. In conclusion, pDC-AML is characterized by a high frequency of RUNX1 mutations and increased expression of a pDC transcriptional program. CD123 targeting represents a potential treatment approach for pDC-AML.

Azacitidine + venetoclax, decitabine + venetoclax, and low-dose cytarabine + venetoclax are now standard treatments for newly diagnosed older or unfit patients with acute myeloid leukemia (AML). Although these combinations are also commonly used in relapsed or refractory AML (RR-AML), clinical and molecular predictors of response and survival in RR-AML are incompletely understood. We retrospectively analyzed clinical and molecular characteristics and outcomes for 86 patients with RR-AML who were treated with venetoclax combinations. The complete remission (CR) or CR with incomplete hematologic recovery (CRi) rate was 24%, and the overall response rate was 31% with the inclusion of a morphologic leukemia-free state. Azacitidine + venetoclax resulted in higher response rates compared with low-dose cytarabine + venetoclax (49% vs 15%; P = .008). Median overall survival (OS) was 6.1 months, but it was significantly longer with azacitidine + venetoclax compared with low-dose cytarabine + venetoclax (25 vs 3.9 months; P = .003). This survival advantage of azacitidine + venetoclax over low-dose cytarabine + venetoclax persisted when patients were censored for subsequent allogeneic stem cell transplantation (8.1 vs 3.9 months; P = .035). Mutations in NPM1 were associated with higher response rates, whereas adverse cytogenetics and mutations in TP53, KRAS/NRAS, and SF3B1 were associated with worse OS. Relapse was driven by diverse mechanisms, including acquisition of novel mutations and an increase in cytogenetic complexity. Venetoclax combination therapy is effective in many patients with RR-AML, and pretreatment molecular characteristics may predict outcomes. Trials that evaluate novel agents in combination with venetoclax therapy in patients with RR-AML that have adverse risk genomic features are warranted.

We investigated the role of PRMT5 in myeloproliferative neoplasm (MPN) pathogenesis and aimed to elucidate key PRMT5 targets contributing to MPN maintenance. PRMT5 is overexpressed in primary MPN cells, and PRMT5 inhibition potently reduced MPN cell proliferation ex vivo. PRMT5 inhibition was efficacious at reversing elevated hematocrit, leukocytosis, and splenomegaly in a model of JAK2V617F+ polycythemia vera and leukocyte and platelet counts, hepatosplenomegaly, and fibrosis in the MPLW515L model of myelofibrosis. Dual targeting of JAK and PRMT5 was superior to JAK or PRMT5 inhibitor monotherapy, further decreasing elevated counts and extramedullary hematopoiesis in vivo. PRMT5 inhibition reduced expression of E2F targets and altered the methylation status of E2F1 leading to attenuated DNA damage repair, cell-cycle arrest, and increased apoptosis. Our data link PRMT5 to E2F1 regulatory function and MPN cell survival and provide a strong mechanistic rationale for clinical trials of PRMT5 inhibitors in MPN. Significance: Expression of PRMT5 and E2F targets is increased in JAK2V617F+ MPN. Pharmacologic inhibition of PRMT5 alters the methylation status of E2F1 and shows efficacy in JAK2V617F/MPLW515L MPN models and primary samples. PRMT5 represents a potential novel therapeutic target for MPN, which is now being clinically evaluated. This article is highlighted in the In This Issue feature, p. 1611

The role of allogeneic hematopoietic cell transplant (allo-HCT) as consolidation after initial venetoclax therapy and the efficacy of venetoclax salvage therapy for relapse after allo-HCT in patients with acute myeloid leukemia (AML) are unclear. We conducted a retrospective study of patients with AML or myelodysplastic syndrome (MDS) who received venetoclax either before or after allo-HCT at Memorial Sloan Kettering Cancer Center and Yale University from 11 August 2016 to 16 November 2020. Among 39 heavily pretreated patients who received venetoclax before allo-HCT, median OS from allo-HCT was not reached after a median follow up of 12.5 months resulting in a 12-month OS estimate of 79.0%. In 37 patients who had received venetoclax-based combinations as salvage therapy after allo-HCT, the overall response rate was 32% with a median OS of 4.7 months (12-month OS estimate: 43.4%). Four patients underwent a second allo-HCT following venetoclax-based salvage therapy suggesting it as a potential salvage treatment option.

Approximately 15% of chronic lymphocytic leukemia/small lymphocytic lymphomas (CLL/SLL) transform into aggressive lymphomas, often diffuse large B cell lymphomas (DLBCL), known as Richter Syndrome ...

Increasing evidence supports the prognostic significance of measurable residual disease (MRD) in acute myeloid leukemia (AML). Dynamic MRD assessment for patients with AML complements baseline patient risk assessment factors in determining patient prognosis. MRD status may also be helpful in informing therapeutic decisions. The European Leukemia Net MRD working party recently issued consensus recommendations for the use of MRD in AML. The Food and Drug Administration also issued advice for using MRD in trials of hematologic malignancies. This article discusses MRD testing, highlights the challenges in adopting MRD testing in clinical practice, and provides insights into the future of the field.

Plasmacytoid dendritic cells (pDC) are the principal natural type I interferon producing dendritic cells. Neoplastic expansion of pDCs and pDC precursors leads to blastic plasmacytoid dendritic cell neoplasm (BPDCN) and clonal expansion of mature pDCs has been described in chronic myelomonocytic leukemia (CMML). The role of pDC expansion in acute myeloid leukemia (AML) is poorly studied. Here we characterize AML patients with pDC expansion (pDC-AML), which we observe in approximately 5% of AML. pDC-AML often possess crosslineage antigen expression and have adverse risk stratification with poor outcome. RUNX1 mutations are the most common somatic alterations in pDC-AML (>70%) and are much more common than in AML without PDC expansion. We demonstrate that pDCs are clonally related to, and originate from, leukemic blasts in pDC-AML. We further demonstrate that leukemic blasts from RUNX1-mutated AML upregulate a pDC transcriptional program, poising the cells towards pDC differentiation and expansion. Finally, tagraxofusp, a targeted therapy directed to CD123, reduces leukemic burden and eliminates pDCs in a patient-derived xenograft model. In conclusion, pDC-AML is characterized by a high frequency of RUNX1 mutations and increased expression of a pDC transcriptional program. CD123 targeting represents a potential treatment approach for pDC-AML.

In patients with multiple myeloma, plasmablastic transformation in the bone marrow is rare and associated with poor outcomes. The significance of discordant extramedullary plasmablastic transformation in patients with small, mature clonal plasma cells in the bone marrow has not been well studied. Here, we report the clinicopathologic, cytogenetic, and molecular features of 10 such patients (male/female: 6/4, median age: 65 y, range: 48 to 76 y) with an established diagnosis of multiple myeloma in the bone marrow composed of small, mature plasma cells in parallel with a concurrent or subsequent extramedullary plasmablastic transformation. Eight patients with available survival data showed an overall aggressive clinical course with a median survival of 4.5 months after the diagnosis of extramedullary plasmablastic transformation, despite aggressive treatment and even in patients with low-level bone marrow involvement. Pathologically, the extramedullary plasmablastic myeloma were clonally related to the corresponding bone marrow plasma cells, showed high levels of CMYC and/or P53 expression with a high Ki-67 proliferation index by immunohistochemistry and harbored more complex genomic aberrations including frequent mutations in the RAS pathway and MYC rearrangements compared with their bone marrow counterparts. In summary, although genetic and immunohistochemical studies were not uniformly performed on all cases due to the retrospective nature of this study, our data suggest that discordant extramedullary plasmablastic transformation of multiple myeloma has an aggressive clinical course and is characterized by frequent mutations in the RAS pathway and more complex genomic abnormalities.

TET2 and DNMT3A mutations are frequently identified in T-cell lymphomas of T follicular helper cell origin (TCL-TFH), clonal hematopoiesis (CH), and myeloid neoplasms (MNs). The relationships among these 3 entities, however, are not well understood. We performed comprehensive genomic studies on paired bone marrow and tissue samples as well as on flow cytometry–sorted bone marrow and peripheral blood subpopulations from a cohort of 22 patients with TCL-TFH to identify shared CH-type mutations in various hematopoietic cell compartments. Identical mutations were detected in the neoplastic T-cell and myeloid compartments of 15 out of 22 patients (68%), including TET2 (14/15) and DNMT3A (10/15). Four patients developed MNs, all of which shared CH-type mutations with their TCL-TFH; additional unique genetic alterations were also detected in each patient’s TCL-TFH and MN. These data demonstrate that CH is prevalent in patients with TCL-TFH and that divergent evolution of a CH clone may give rise to both TCL-TFH and MNs.

Key Points Core-binding factor AML can evolve from good-risk disease into aggressive disease through the gain of additional genomic aberrations. In this unique case, an AML patient died of hypereosinophilic syndrome with solid organ infiltration of differentiated eosinophils.

Genomic studies in acute myeloid leukemia (AML) have generated a near complete catalogue of genes mutated at varying frequencies both across patients and in individual leukemias. The high variability of mutation burden within a given leukemia is suggestive of a stepwise evolutionary process composed of early, clonal, mutations and subsequent subclonal events. The receptor tyrosine kinase, FLT3, is the most commonly mutated gene in AML, with mutations frequently manifesting as internal tandem duplications (ITDs) in the juxtamembrane domain leading to constitutive kinase activation. Although FLT3 is commonly a subclonal mutational event, FLT3 ITD mutations portend a poor prognosis particularly when combined with DNMT3A and NPM1, earlier mutations that drive clonal expansion. Notwithstanding its role as a subclonal driver, previous preclinical FLT3 models have utilized retroviral overexpression or germline mutant expression at the endogenous locus precluding accurate temporal modeling of disease. These efforts have prohibited evaluation of FLT3 mutational acquisition in the context observed in AML patients. Here, we report the development of an endogenously targeted, Flp inducible, Flt3 ITD mouse allele which can be somatically activated subsequent to cooperating disease alleles. When activated with a tamoxifen inducible FlpoER, Flt3 mutant mice developed rapid leukocytosis peaking at 4-6 weeks post activation and resolving by 8-10 weeks, a finding not previously observed in constitutive models. This leukocytosis was disproportionately monocytic and accompanied by pronounced anemia and thrombocytopenia. Long term, these mice develop a myeloproliferative disease , reminiscent of previously reported constitutive alleles. In competitive transplantation studies, Flt3 mutant cells initiated disease and outcompeted wild-type cells. Despite this competitive advantage, disease was incapable of transplanting into secondary recipients. We further observed a non-cell autonomous depletion of SLAM+ LSKs suggesting the Flt3 mutant cells cannot propagate disease in self-renewing stem cells. To evaluate how this allele influenced leukemic evolution we crossed this Flt3 ITD allele to a Flp inducible Npm1 c mouse where a pulse of tamoxifen simultaneously activated both alleles. The combination of mutant Npm1 and Flt3 resulted in progressive leukocytosis which did not resolve. Within 6 weeks of mutational activation, these mice developed a lethal AML with robust anemia, thrombocytopenia, leukocytosis and expanded cKIT+ blasts in the blood. RNA-sequencing and immunophenotyping by CyTOF revealed distinct patterns of differentiation, gene-expression and downstream signaling.In an effort to model sequential mutational acquisition, we crossed the Flp Flt3 ITD allele to a Cre-inducible Dnmt3a R878H. Cre mRNA was electroporated into lineage negative bone marrow cells to activate the Dnmt3a R878H allele and transplanted into lethally irradiated recipients. Four weeks post engraftment, Flt3 ITD was activated with a pulse of tamoxifen. In contrast to the Flt3-Npm1 model, we observed an increase and subsequent decrease in WBC similar to the kinetics observed in Flt3 ITD only mice. However, by 20 weeks we observed a robust and consistent increase in WBC accompanied by an emergence of cKIT+ cells in the blood. Histopathology indicated that >50% of mice expressing both alleles in sequence developed AML marked by increased blasts in the marrow, with moderate anemia and thrombocytopenia compared to the Flt3-Npm1 models. Critically, in contrast to Flt3 ITD only mice, acquisition of the Flt3 ITD in Npm1 or Dnmt3a mutant HPSCs induced fully transplantable AML with immunophenotypic characteristics seen in human AML with these same genotypes. Collectively these results demonstrate that different co-occurring mutations are capable of transforming Flt3 ITD mutant cells, albeit with distinct latencies and mechanisms of cooperativity. In summary, our studies utilizing novel multi-recombinase models of leukemogenesis reveal new insights into the early phase of oncogene activation, and how cooperating alleles influence this response. This inducible Flt3 ITD allele represents a significant advance in modeling clonal evolution in myeloid malignancies and provides a critical isogenic platform for preclinical development of novel leukemia therapeutic regimens. Figure 1 Figure 1. Disclosures Bowman: Mission Bio: Honoraria, Speakers Bureau. Xiao: Stemline Therapeutics: Research Funding. Miles: Mission Bio: Honoraria, Speakers Bureau. Trowbridge: Fate Therapeutics: Patents & Royalties; H3 Biomedicine: Research Funding. Levine: Amgen: Honoraria; Lilly: Honoraria; Mission Bio: Membership on an entity's Board of Directors or advisory committees; Imago: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Ajax: Membership on an entity's Board of Directors or advisory committees; QIAGEN: Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria; Zentalis: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Research Funding; Janssen: Consultancy; Astellas: Consultancy; Morphosys: Consultancy; Incyte: Consultancy; Auron: Membership on an entity's Board of Directors or advisory committees; Prelude: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Background CD19-targeted chimeric-antigen receptor-modified T-cells (CAR-T) are promising in the treatment of refractory B-lymphoblastic leukemia (B-ALL). Minimal residual disease (MRD) detection by multicolor flow cytometry (FCM) is critical to distinguish B-ALL MRD from regenerating, non-neoplastic B-cell populations. Methods FCM was performed on samples from 9 patients with B-ALL treated with CAR-T. Results All 9 patients showed response to CAR-T. Additionally, FCM revealed circulating CD10 + B cells, potentially mimicking MRD. Circulating CD10+ B-cells were detected in blood from 3 days to 3 months after CAR-T, comprising 73% (median) of B-cells (52–83%, 95%CI). They expressed CD19, CD10, CD20, bright CD9, CD22, CD24, moderate CD38 and dim CD58, but were CD34 (–), with bright CD45 and polyclonal surface light chain immunoglobulin (sIg) expression. A similar CD10 + B-cell subpopulation was detected by marrow FCM, amidst abundant B-cell precursors. Conclusions These circulating CD10 + B-cells are compatible with immature B-cells, and are a reflection of B-cell recovery within the marrow. They are immunophenotypically distinguishable from residual B-ALL. Expression of light chain sIg and key surface antigens characterizing regenerating B-cell precursors can distinguish immature B-cells from B-ALL MRD and prevent misdiagnosis. © 2017 International Clinical Cytometry Society

While the majority of NK/T-cell neoplasms are unfavorable in clinical outcome, a few pathologic variants demonstrate relatively indolent behavior. These include primarily four types of tissue-based T-cell or NK-cell lymphoproliferative disorders and two leukemic forms of T-cell or NK-cell lymphoproliferative disorder. The diagnostic criteria and other clinicopathologic features of these entities are discussed as question and answer format.

TAFRO syndrome, a clinical subtype of idiopathic multicentric Castleman disease (iMCD), consists of a constellation of symptoms/signs including thrombocytopenia, anasarca, fever, reticulin fibrosis/renal dysfunction, and organomegaly. The etiology of iMCD-TAFRO and the basis for cytokine hypersecretion commonly seen in iMCD-TAFRO patients has not been elucidated. Here, we identified a somatic MEK2P128L mutation and a germline RUNX1G60C mutation in two patients with iMCD-TAFRO, respectively. The MEK2P128L mutation, which has been identified previously in solid tumor and histiocytosis patients, caused hyperactivated MAP kinase signaling, conferred IL-3 hypersensitivity and sensitized the cells to various MEK inhibitors. The RUNX1G60C mutation abolished the transcriptional activity of wild-type RUNX1 and functioned as a dominant negative form of RUNX1, resulting in enhanced self-renewal activity in hematopoietic stem/progenitor cells. Interestingly, ERK was heavily activated in both patients, highlighting a potential role for activation of MAPK signaling in iMCD-TAFRO pathogenesis and a rationale for exploring inhibition of the MAPK pathway as a therapy for iMCD-TAFRO. Moreover, these data suggest that iMCD-TAFRO might share pathogenetic features with clonal inflammatory disorders bearing MEK and RUNX1 mutations such as histiocytoses and myeloid neoplasms.

Hodgkin/Reed-Sternberg (HRS) cells in the setting of chronic lymphocytic leukemia (CLL) exist in 2 forms: type I with isolated HRS cells in a CLL background (Hodgkin-like lesion) and type II with typical classic Hodgkin lymphoma, a variant of Richter transformation (CHL-RT). The clinical significance of the 2 morphological patterns is unclear, and their biological features have not been compared. We retrospectively reviewed 77 cases: 26 of type I and 51 of type II CHL-RT; 3 cases progressed from type I to type II. We examined clinical features, Epstein-Barr virus (EBV) status, and clonal relatedness after microdissection. Median age for type I was 62 years versus 73 years for type II (P=.01); 27% (type I) versus 73% (type II) had a history of CLL. HRS cells were positive for EBV in 71% (55/77), similar in types I and II. Clonality analysis was performed in 33 cases (type I and type II combined): HRS cells were clonally related to the underlying CLL in 14 and unrelated in 19. ZAP-70 expression of the CLL cells but not EBV status or morphological pattern was correlated with clonal relatedness: all 14 clonally related cases were ZAP-70 negative, whereas 74% (14/19) of clonally unrelated cases were ZAP-70 positive. Overall median survival (types I and II) after diagnosis was 44 months. Advanced age was an adverse risk factor for survival, but not histologic pattern, type I versus type II. HRS-like cells in a background of CLL carries a similar clinical risk to that of CHL-RT and may progress to classic Hodgkin lymphoma in some cases.

Measurable residual disease is associated with inferior outcomes in patients with acute myeloid leukemia (AML). Measurable residual disease monitoring enhances risk stratification and may guide therapeutic intervention. The European LeukemiaNet working party recently came to a consensus recommendation incorporating leukemia associated immunophenotype-based different from normal approach by multi-color flow cytometry for measurable residual disease evaluation. However, the analytical approach is highly expertise-dependent and difficult to standardize. Here we demonstrate that loss of plasmacytoid dendritic cell differentiation after 7+3 induction in AML is highly specific for measurable residual disease positivity (specificity 97.4%) in a uniformly treated patient cohort. Moreover, loss of plasmacytoid dendritic cell differentiation as determined by a blast-to-plasmacytoid dendritic cell ratio >10 was strongly associated with inferior overall and relapse-free survival (RFS) [Hazard ratio 2.79, 95% confidence interval (95%CI): 0.98-7.97; P=0.077) and 3.83 (95%CI: 1.51-9.74; P=0.007), respectively), which is similar in magnitude to measurable residual disease positivity. Importantly, measurable residual disease positive patients who reconstituted plasmacytoid dendritic cell differentiation (blast/ plasmacytoid dendritic cell ratio

Traditionally low-grade B-cell lymphomas have been excluded from the category of monomorphic posttransplant lymphoproliferative disorders. However, recent reports identified Epstein-Barr virus-positive (EBV) extranodal marginal zone lymphomas (MZL), almost exclusively seen in the posttransplant setting. Some reported cases responded to reduced immunosuppression, suggesting that they should be considered as a form of posttransplant lymphoproliferative disorders. We identified 10 cases of EBV MZL, 9 in extranodal sites and 1 presenting in lymph node. Two cases arose following solid organ transplantation, but other settings included iatrogenic immunosuppression for rheumatoid arthritis (2); prior chemotherapy (2); congenital immune deficiency (1); and increased age (3), as the only potential cause of immune dysfunction. There were 4 males and 6 females; age range 18 to 86. The atypical plasmacytoid and/or monocytoid B cells were positive for EBV in all cases, with either latency I or II in all cases tested. Monotypic light chain expression was shown in all with 6 cases positive for IgG, and 2 for IgM, undetermined in 2. Clonal immunoglobulin gene rearrangement was positive in all cases with successful amplification. MYD88 L265P was wild type in the 6 cases tested. We show that EBV MZLs can arise in a variety of clinical settings, and are most often extranodal. Treatment varied, but most patients had clinically indolent disease with response to reduction of immune suppression, or immunochemotherapy.

The B/T subtype of mixed phenotype acute leukemia (B/T MPAL) is defined by co-expression of antigens of both B- and T-cell lineages on leukemic blasts. Although it has been suggested that multilineage antigen expression portends poor response to chemotherapy, the clinical characteristics and driver mutations that underlie the pathogenesis of this rare subtype of acute leukemia are scarcely known. We identified nine cases of B/T MPAL from multiple institutions and correlated clinical and immunophenotypic findings with next-generation sequencing data. We report that B/T MPAL commonly presents with lymphadenopathy in adolescence and young adulthood. While the tumors have diverse cytogenetic and genomic perturbations, recurrent acquired aberrations include mutations in the putative transcriptional regulator PHF6 and the JAK-STAT and Ras signaling pathways. Alterations were also identified in genes encoding hematopoietic transcription factors, cell cycle regulators/tumor suppressors, and chromatin modifying enzymes. The genomic landscape of B/T MPAL strongly resembles that of T-ALL subgroups associated with early developmental arrest, while genetic alterations that are common in B-ALL were rarely seen. Two-thirds of the patients responded to ALL-based chemotherapy with or without stem cell transplantation. Our observations lay the groundwork for further study of the unique biology and clinical trajectory of B/T MPAL.

Genomic analysis has greatly influenced the diagnosis and clinical management of patients affected by diverse forms of hematologic malignancies. Here, we review how genetic alterations define subclasses of patients with acute leukemias, myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPNs), non-Hodgkin lymphomas, and classical Hodgkin lymphoma. These include new subtypes of acute myeloid leukemia defined by mutations in RUNX1 or BCR-ABL1 translocations as well as a constellation of somatic structural DNA alterations in acute lymphoblastic leukemia. Among patients with MDS, detection of mutations in SF3B1 define a subgroup of patients with the ring sideroblast form of MDS and a favorable prognosis. For patients with MPNs, detection of the BCR-ABL1 fusion delineates chronic myeloid leukemia from classic BCR-ABL1− MPNs, which are largely defined by mutations in JAK2, CALR, or MPL. In the B-cell lymphomas, detection of characteristic rearrangements involving MYC in Burkitt lymphoma, BCL2 in follicular lymphoma, and MYC/BCL2/BCL6 in high-grade B-cell lymphomas are essential for diagnosis. In T-cell lymphomas, anaplastic large-cell lymphoma is defined by mutually exclusive rearrangements of ALK, DUSP22/IRF4, and TP63. Genetic alterations affecting TP53 and the mutational status of the immunoglobulin heavy-chain variable region are important in clinical management of chronic lymphocytic leukemia. Additionally, detection of BRAFV600E mutations is helpful in the diagnosis of classical hairy cell leukemia and a number of histiocytic neoplasms. Numerous additional examples provided here demonstrate how clinical evaluation of genomic alterations have refined classification of myeloid neoplasms and major forms of lymphomas arising from B, T, or natural killer cells.

TAFRO syndrome is a rare clinicopathologic variant of idiopathic multicentric Castleman disease characterized by Thrombocytopenia, Ascites (anasarca), myeloFibrosis, Renal dysfunction, and Organomegaly. Here, we report a case of TAFRO syndrome in an HIV-negative young Caucasian male who presented with fever, normocytic anemia, thrombocytopenia, and acute renal insufficiency. The serum interleukin-6 (IL-6) level was elevated. Chest and abdominal CT revealed bilateral pleural effusion, ascites, splenomegaly, and multiple mildly enlarged lymph nodes. An excisional biopsy of inguinal lymph node showed a few atrophic follicles and expansion of interfollicular areas by marked vascular proliferation and polytypic plasmacytosis. HHV-8 was negative. Subsequent bone marrow biopsy was normocellular with moderately increased megakaryocytes and occasional megakaryocytic emperipolesis. His signs and symptoms improved after treatment with methylprednisolone and tocilizumab (anti-IL-6 receptor antibody). Our study confirms the distinctive nature of this syndrome, which should allow for better recognition and appropriate therapy. Keywords: Castleman-Kojima disease, TAFRO syndrome, Idiopathic multicentric Castleman disease, IL-6, HHV-8

Background Thrombotic thrombocytopenic purpura (TTP) is a life-threatening diagnosis requiring prompt initiation of therapeutic plasma exchange (TPE). Measurement of immature platelet (PLT) fraction (%-IPF) differentiates PLT consumption or destruction from hypoproduction. Study Design and Method Our study evaluated %-IPF changes over the course of TTP treated with TPE and as a measure of treatment efficacy. Eleven idiopathic TTP patients, two human immunodeficiency virus (HIV)-associated TTP patients, and five non-TTP patients with thrombocytopenia were enrolled into our study. All patients were treated with TPE and had ADAMTS13 activity measured. Results All idiopathic TTP patients had a significantly increased %-IPF and decreased absolute immature PLT count (A-IPC) and PLT count at presentation. An A-IPC value of less than 5 × 109/L at presentation has 84.6% sensitivity, 80% specificity, and 91.7% positive predictive value for diagnosing TTP. A concurrent steady decline in %-IPF and increased PLT counts toward normal was observed in TTP patients undergoing TPE. The A-IPC, however, showed an increase and decrease curve that was not seen in the two HIV-associated TTP patients with no response to TPE and the five non-TTP patients. More importantly, reaching an A-IPC ratio of 3 compared to baseline value during TPE can readily differentiate idiopathic TTP from the other two groups and is correlated with good clinical responses to TPE. An abrupt increase of A-IPC during TPE was also noted in a TTP patient who relapsed 3 days before PLT count decrease. A-IPC is positively correlated with ADAMTS13 activity at presentation but negatively correlated with ADAMTS13 activity during recovery. Conclusion A-IPC should be routinely analyzed for diagnosing and monitoring TTP patients.

CRABP-II, a retinoic acid binding protein, shuffles retinoic acid from cytoplasm into nucleus and forms a complex with nuclear retinoic acid receptor to facilitate transcriptional activities of retinoic acid. In this study, we studied the expression patterns of CRABP-II in pancreatic ductal adenocarcinoma (PDAC) compared with those in normal pancreas, chronic pancreatitis, and precancerous lesions. We showed no detectable expressions of CRABP-II in normal pancreatic parenchyma, normal ductal epithelium, and chronic pancreatitis. In contrast, the expression of CRABP-II was readily detected in all PDACs including metastatic PDACs. CRABP-II staining was also observed and progressively increased from pancreatic intraepithelial neoplasia 1 to 3. In addition, when fine needle aspiration specimens were evaluated from patients with PDAC, CRABP-II was positive in 55.6% cases if cytology diagnosis was "atypia," and in 87.5% cases, if "malignancy." Our study suggests that CRABP-II is highly and specifically expressed in PDAC and is more commonly expressed in high-grade precursor cancerous lesions than in low-grade lesions. Therefore, overexpression of CRABP-II is a late event of pancreatic carcinogenesis, and it could be used as a diagnostic marker to distinguish PDAC from other benign pancreatic conditions in both resection and cytology specimens.

Background: Myeloid and lymphoid leukemias each have distinct diagnostic algorithms, treatment paradigms, and therapeutic options. In mixed phenotype acute leukemia (MPAL), myeloid and lymphoid immunophenotypes are expressed simultaneously. MPAL is rare, accounting for fewer than 5% of all leukemias, and carries a poor prognosis. Currently, there is no standard of care for treatment of this disease and therapeutic options tailored to this group of patients are lacking. Additionally, the biological mechanisms underlying lineage infidelity are poorly understood. Here we seek to establish more precise biological characterization of this disease and have identified a new subgroup of patients with Mixed Phenotype Acute Myeloid Leukemia (AML-MP). Methods: We developed a novel natural language processing pipeline and used it to review 830 patient records, representing all acute leukemia patients treated at Memorial Sloan Kettering Cancer Center since 2015. We identified those with ambiguous lineages based on multiparameter flow cytometry data and integrated next-generation sequencing, cytogenetics, and clinical information. Statistical methods are outlined with the associated results below. Results: Among the 830 patient records reviewed, 54 (6.5%) patients with mixed lineage characteristics were identified. Of these, 26 (48%) carried a formal diagnosis of MPAL while 28 (52%) carried a diagnosis of AML with myelodysplasia related changes (AML-MRC) or therapy related AML (t-AML). Among the cases expressing multiple lineages, 34 (25%) had B/Myeloid features (11 MPAL, 23 AML-MRC), 17 (13%) had T/Myeloid features (13 MPAL, 4 AML-MRC), 3 (0.7%) had B/T/Myeloid lineage (2 MPAL, 1 AML-MRC). Only 8 patients received prior chemotherapy at our institution > 1 year prior to diagnosis and were classified as t-AML. As a control group, we also identified 79 patients with AML-MRC exhibiting myeloid lineage alone. We pooled the mixed-lineage and myeloid-only AML-MRC cases and performed k-means clustering into 2 groups using all available molecular features (Figure 1a). Although TP53 was enriched within AML cases (p Among patients with mixed lineage characteristics, we performed a cox proportional hazards analysis and found that RUNX1 and SRSF2 mutations were predictors of better overall survival (OS) after adjusting for age, cytogenetics, and diagnosis type (age > 60: p < 0.01, high risk cytogenetics: p Among AML-MRC myeloid only cases, ASXL1 mutation was associated with improved OS and TET2 mutation showed a trend toward poorer OS after adjusting for age and cytogenetic risk (age>60: p=0.01, ASXL1: p=0.02, TET2: p=0.06; overall: p < 0.01, Figure 1b, right). Cytogenetic risk did not independently contribute to the model (high risk: p=0.3, intermediate risk: p=0.55). We also manually reviewed flow cytometry results from 64 patients diagnosed with MPAL or diagnosed with t-AML or AML-MRC despite expressing markers of multiple lineages (Mixed Phenotype AML). Using a k-nearest neighbor approach, we found that immunophenotype identified two distinct patient populations - one largely Mixed Phenotype AML, and the other with a mixture of MPAL and Mixed Phenotype AML cases (p Conclusions: Genomic characteristics improve prognostication for patients with MPAL, with RUNX1 and SRSF2 predicting a more favorable OS and TP53 not predictive of worse OS; this is in contrast to current stratifications of patients with de novo AML. While TP53 is enriched among AML cases, it does not distinguish AML-MRC from MPAL. Additionally, AML-MRC patients with ASXL1 mutations had improved overall survival after adjusting for cytogenetic risk. Some cases that are categorized as AML-MRC and t-AML by WHO criteria should be considered Mixed Phenotype AML and may be better classified into one of two distinct immunophenotypic subsets. Our study and model provide a more refined biological classification and prognostic schema for patients with MPAL and AML-MP; further validation of these observations is required in other data sets. Figure 1 Disclosures Roshal: Auron Therapeutics: Equity Ownership, Other: Provision of services; Celgene: Other: Provision of Services; Physicians' Education Resource: Other: Provision of services. Levine:Prelude Therapeutics: Research Funding; Roche: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Gilead: Consultancy; Qiagen: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Loxo: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Imago Biosciences: Membership on an entity's Board of Directors or advisory committees; Lilly: Honoraria; Amgen: Honoraria. Tallman:Indy Hematology Review: Honoraria; Hematology Oncology of Indiana: Honoraria; Salzberg Weill Cornall MSKCC Seminar in Hematologic Malignancies: Honoraria; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; International Conference in Leukemia: Honoraria; 14th Annual Miami Cancer Meeting: Honoraria; Amgen: Consultancy; Orsenix: Membership on an entity's Board of Directors or advisory committees, Research Funding; UpToDate: Patents & Royalties; Mayo Clinic: Honoraria; New Orleans Summer Cancer Conference: Honoraria; ADC Therapeutics: Research Funding; Danbury Hospital Tumor Board: Honoraria; Arog Pharmaceuticals: Research Funding; Nohla: Membership on an entity's Board of Directors or advisory committees, Research Funding; KAHR: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Membership on an entity's Board of Directors or advisory committees; BioSight: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bioline: Membership on an entity's Board of Directors or advisory committees, Research Funding; University of Oklahoma Medical Center: Honoraria; Cellerant Therapeutics: Research Funding; Rigel: Membership on an entity's Board of Directors or advisory committees.

Background Myelodysplastic syndrome (MDS) is a clonal, pre-leukemic stem cell disorder characterized by decreased blood counts due to ineffective hematopoiesis. Plasmacytoid dendritic cells (PDC) are stem cell derived, type I interferon producing dendritic cells, that are readily identifiable by flow cytometry (FC) using expression of CD123 and HLA-DR. PDCs have been shown to be decreased in acute myeloid leukemia (AML). Our study uses FC to evaluate PDC in MDS, their relationship to the Revised International Prognostic Scoring System (IPSS-R) for MDS, as well as their relationship to clinical outcomes. Methods We identified 197 patients with new MDS diagnoses and examined FC data of bone marrow aspirates (BMA) at first presentation to our institution for blast and PDC percentages. Patients who presented with an outside diagnosis of MDS greater than 1 year before presentation to our institution were excluded. MDS with excess blasts 1 and 2 (MDS-EB1/EB2) were designated as high grade, while MDS with isolated del5q (MDS-d5q), single lineage dysplasia (MDS-SLD), and multilineage dysplasia (MDS-MLD) were designated as low grade, with or without ring sideroblasts. 163 patients had sufficient data to calculate their IPSS-R risk categories. Sixteen patients with a history of solid tumor malignancies undergoing BMA for cytopenias were used as controls. The bone marrow of these control patients showed no evidence of morphologic dysplasia, they had normal karyotypes, and no pathogenic variants were detected on a 28 gene next generation sequencing based myeloid panel. We used CD34 and CD117 to quantify blasts, and CD123 and HLA-DR to quantify PDCs. Results The proportion of PDCs expressed as a percentage of total WBCs was significantly lower in higher risk MDS diagnoses (p Discussion This study demonstrates that PDC proportions decrease with MDS disease progression and are progressively lower as IPSS-R risk category increases. We also demonstrate that quantification of PDC in MDS can aid in predicting outcomes, although this may be due to a strong association with IPSS-R categories. FC is a useful and clinically feasible tool for quantitating PDCs in bone marrow aspirates, and this measurement is correlated with risk in MDS. Disclosures Arcila: Invivoscribe, Inc.: Consultancy, Honoraria. Roshal:Celgene: Other: Provision of Services; Auron Therapeutics: Equity Ownership, Other: Provision of services; Physicians' Education Resource: Other: Provision of services.

Clonal heterogeneity and evolution of mantle cell lymphoma (MCL) remain unclear despite the progress in our understanding of its biology. Here, we report a 71-yr-old male patient with an aggressive MCL and depict the clonal evolution from initial diagnosis of typical MCL to relapsed blastoid MCL. During the course of the disease, the patient was diagnosed with classic Hodgkin lymphoma (CHL) and received a CHL therapeutic regimen. Molecular analysis by next-generation sequencing of both MCL and CHL demonstrated clonally related CHL with characteristic immunophenotype and PDL1/2 gains. Moreover, our data illustrate the clonal heterogeneity and acquisition of additional genetic aberrations including a rare fusion of SEC22B-NOTCH2 in the process of clonal evolution. Evidence obtained from our comprehensive immunophenotypic and genetic studies indicates that MCL and CHL can originate from a common precursor by divergent clonal evolution, which may pose a therapeutic challenge.

Introduction Secondary acute myeloid leukemia (s-AML) includes AML with myelodysplasia-related changes (AML-MRC) transformed from an antecedent diagnosis of myelodysplastic syndrome (MDS) or from myeloproliferative and myelodysplastic overlap syndrome (MPN/MDS), as well as therapy-related AML (t-AML) arising in patients with prior exposure to leukemogenic therapies. S-AML has a dismal prognosis. Although the genomic profiling of AML has been well studied, few studies have focused on the molecular aberrations of s-AML. A recent study has demonstrated shared genomic aberrations by AML-MRC and t-AML including mutations in TP53, SRSF2, SF3B1, U2AF1, ZRSR2, ASXL1, EZH2, BCOR, or STAG2. Another recent study has shown that AML patients with mutations in TP53 and chromatin-splicesosome confer poor prognosis and likely represent s-AML. As such, further characterization of the genomic landscape of s-AML is needed. Recurrent somatic PHF6 mutations are rare in de novo AML (~3%), but the frequency and characteristics of PHF6 mutated s-AML are largely unknown. Patients and Methods AML-MRC and t-AML patients were searched from the patient database at the MSKCC between 1/2014 and 12/2016. All the patients were reviewed and the diagnosis was confirmed. Only patients with genomic sequencing studies (a targeted NGS panel comprising 30 genes with mutations enriched in AML) performed at diagnosis were included. Results 152 patients were identified. There were 7 t-AML patients with favorable cytogenetic abnormalities that likely represent a distinct biological subgroup; therefore these cases were excluded from further analysis. AML-MRC (N=75) and t-AML (N=70) patients had similar clinical characteristics including ages, gender distribution, cytogenetic risk, treatment and survival (median survival: 8 months in t-AML and 10 months in AML-MRC, p=0.4); The mutational profiles of AML-MRC and t-AML cohorts were also comparable with recurrent TP53, PHF6, RAS, and RUNX1 mutations, albeit with different frequencies (Figure 1; Red represents t-AML and blue AML-MRC). Therefore we combined AML-MRC and t-AML cohorts for further analysis. 43 (29.7%) patients had TP53 mutations, 17 (11.7%) patients had PHF6 mutations, and 85 (58.6%) patients had neither mutation (non-TP53/non-PHF6). TP53 and PHF6 mutations were mutually exclusive. TP53 mutations were nearly exclusively associated with adverse cytogenetic abnormalities including alterations involving chromosomes (chr) 5p and/or 7q (41/43, 95.3%) with the majority being complex karyotype (30/43, 70%). By contrast, only 18% PHF6 mutated s-AML patients had chr 5p/7q abnormalities and none of them had complex karyotype. Among the patients with non-TP53/non-PHF6 mutations, 30% had chr 5p/7q abnormalities and 6% had complex karyotype. Although secondary AML has very poor prognosis, PHF6 mutated patients had better overall survival than the other two groups (Log rank test, p=0.0095; median survival: PHF6 vs TP53 vs. non-TP53/non-PHF6: 24 vs. 7 vs. 9 months). Immunophenotypically, the blasts from PHF6 mutated patients showed a high frequency of loss of CD38 (44% vs 17%, p=0.04) and CD33 expression (69% vs 28%, p=0.004) compared to PHF6 unmutated cases, indicative of a less mature, stem-cell derived phenotype Conclusion PHF6 mutations define a unique subset of s-AML that is associated with a more primitive stem/progenitor immunophenotype, absent complex karyotype and relatively better outcomes. PHF6 mutations are mutually exclusive to TP53 mutations. Studies are needed to further define the genomic classification of s-AML patients in an unbiased way and to elucidate the role of PHF6 mutations in s-AML. Figure 1 Figure 1. Disclosures Goldberg: AROG: Research Funding; Abbvie: Research Funding; Pfizer: Research Funding; Celgene: Research Funding. Tallman:Orsenix: Other: Advisory board; AbbVie: Research Funding; Cellerant: Research Funding; BioSight: Other: Advisory board; AROG: Research Funding; Daiichi-Sankyo: Other: Advisory board; ADC Therapeutics: Research Funding. Arcila:Invivoscribe, Inc.: Consultancy, Honoraria. Levine:Loxo: Consultancy, Equity Ownership; Gilead: Honoraria; Roche: Consultancy, Research Funding; Epizyme: Patents & Royalties; Imago: Equity Ownership; Qiagen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; C4 Therapeutics: Equity Ownership; Novartis: Consultancy; Janssen: Consultancy, Honoraria; Isoplexis: Equity Ownership; Prelude: Research Funding.

Given its catalytic activity to generate diacylglycerol and inositol 1,4,5-trisphosphate, phospholipase C (PLC) is implicated in promoting cell growth. However, we found that PLC-beta3-deficient mice develop myeloproliferative disease, lymphoma, and other tumors. The mutant mice have increased numbers of hematopoietic stem cells with increased proliferative, survival, and myeloid-differentiative abilities. These properties are dependent on Stat5 and can be antagonized by the protein phosphatase SHP-1. Stat5-dependent cooperative transformation by active c-Myc and PLC-beta3 deficiency was suggested in mouse lymphomas in PLC-beta3(-/-) and in Emicro-myc;PLC-beta3(+/-) mice and human Burkitt's lymphoma cells. The same mechanism for malignant transformation seems to be operative in other human lymphoid and myeloid malignancies. Thus, PLC-beta3 is likely a tumor suppressor.

Septic transfusion reactions (STRs) resulting from transfusion of bacterially contaminated platelets are a major hazard of platelet transfusion despite recent interventions. Active and passive surveillance for bacterially contaminated platelets was performed over 7 years (2007-2013) by culture of platelet aliquots at time of transfusion and review of reported transfusion reactions. All platelet units had been cultured 24 hours after collection and released as negative. Five sets of STR criteria were evaluated, including recent AABB criteria; sensitivity and specificity of these criteria, as well as detection by active and passive surveillance, were determined. Twenty of 51,440 platelet units transfused (0.004%; 389 per million) were bacterially contaminated by active surveillance and resulted in 5 STRs occurring 9 to 24 hours posttransfusion; none of these STRs had been reported by passive surveillance. STR occurred only in neutropenic patients transfused with high bacterial loads. A total of 284 transfusion reactions (0.55%) were reported by passive surveillance. None of these patients had received contaminated platelets. However, 6 to 93 (2.1%-32.7%) of these 284 reactions met 1 or more STR criteria, and sensitivity of STR criteria varied from 5.1% to 45.5%. These results document the continued occurrence of bacterial contamination of platelets resulting in STR in neutropenic patients, failure of passive surveillance to detect STR, and lack of specificity of STR criteria. These findings highlight the limitations of reported national STR data based on passive surveillance and the need to implement further measures to address this problem such as secondary testing or use of pathogen reduction technologies.

The E2997 Phase III trial included preservation of valuable chronic lymphocytic leukemia (CLL) patient specimens and relevant clinical outcome data. Using a novel high-resolution technology on a flow cytometry platform, we assessed 79 E2997 samples for the expression of 27 analytes that reflected the activity of signaling pathways and apoptosis. We found that the expression levels of ZAP70 segregated the samples into two subpopulations with the distribution showing a peak-trough-peak configuration. Although prior assessment of ZAP70 by standard procedures did not reveal any prognostic information, we found by using the trough in the distribution as a cutpoint that ZAP70 expression levels were significantly correlated with both progression-free survival and overall survival. Additionally, the cells expressing high versus low levels of ZAP70 demonstrated distinct molecular organization as indicated by the other analytes assessed. Our analysis demonstrates the value of ZAP70 expression as a prognostic indicator and suggests that the different clinical results may be due to the distinct molecular biology of the ZAP70-low versus the ZAP70-high CLL samples.

Our aim was to compare rectal perception of patients with diarrhea-predominant irritable bowel syndrome (IBS-D), constipation-predominant irritable bowel syndrome (IBS-C), functional constipation (FC), and healthy controls and to evaluate the therapeutic effect of acupoint transcutaneous electric nerve stimulation (TENS). Age- and sex-matched patients (24 IBS-D, 20 IBS-C, and 30 FC) were selected, and 30 volunteers served as healthy controls. Rectal sensory thresholds were evaluated by rectal balloon distension. Short- and long-term acupoint TENS was given respectively. IBS-D patients had significantly lower rectal sensory thresholds of the first sensation of stool, urgency of defecation, and pain than IBS-C or FC patients or healthy controls (P0.05), but there were no differences in rectal sensory thresholds among IBS-C and FC patients and healthy controls. In each group, females had significantly lower rectal sensory thresholds than males (P0.05), but there was no difference between younger (or = 50 years old) and older (50 years old) patients. Short-term acupoint TENS increased rectal sensory thresholds of IBS-D patients. After 2-month acupoint TENS treatment in IBS-D patients, rectal sensory thresholds were significantly increased, stool times and the intensity of abdominal pain were decreased, and psychological scores were relieved to normal. Lowered rectal perception threshold is a hallmark of IBS-D patients. Females have significantly lower rectal sensory thresholds than males. Acupoint TENS is effective to treat IBS-D.

CDX2, a master transcriptional regulator of intestinal cell differentiation and survival, has been used as a marker to indicate colorectal lineage in adenocarcinomas of unknown origin. Pancreatic ductal adenocarcinoma (PDAC) is one of the most common causes for adenocarcinomas of unknown origin, but CDX2 expression in pancreatic disease remains unclear. In this study, we systemically and extensively investigated the expression and role of CDX2 in PDAC. We reported that CDX2 expression is weak and heterogeneous is all normal pancreas and chronic pancreatitis. It is largely expressed in epithelial-lining cells of pancreatic ducts including main ducts, inter-lobular ducts, intra-lobular ducts, intercalated ducts and centroacinar cells, but not in acinar cells or islet cells. CDX2 expression is down regulated during the transformation process from PanIN to PDAC. Only one third of PDACs retain some degree of CDX2 expression, and this group of PDACs have reduced median survival time compared to that of CDX2 negative group (308 days vs. 586 days, p = 0.0065). Metastatic PDACs remain similar expression pattern to that of the primary sites. Our study clearly demonstrates CDX2 expression in pancreatic diseases including PDAC, which is practically important when CDX2 is used to establish the primary sites of adenocarcinomas of unknown origin. In addition, our study also provides CDX2 as a prognostic marker for PDAC and implicates an important role of CDX2 in the development of normal pancreas and PDAC.

Background ZAP-70 expression has been used as a negative prognostic indicator for patients with chronic lymphocytic leukemia (CLL) (N Engl J Med 2003;348:1764; N Engl J Med 2004;351:893); however, its usefulness as a clinical test has been undermined by technical difficulties relating to the subjective cut-off threshold for positivity. In fact, analyses of samples from CLL patients enrolled in the US Intergroup Phase III trial, E2997, by standard procedures did not result in ZAP-70 (or any other analyte) providing significant prognostic information. We have previously developed a powerful flow cytometric amplification system to assess many important intracytoplasmic molecules including signaling and apoptotic pathway components. We hypothesized that the technology could reveal previously hidden relationships between outcome and molecular expression. Methods We analyzed 79 blood and marrow samples from CLL patients enrolled in the E2997 trial for the expression of ZAP-70 and 28 other analytes. The 28 other analytes included mostly signaling pathway molecules and apoptotic pathway components. Molecular expression levels determined by flow cytometry were analyzed for their relationship to the clinical data. CD19+ B lymphocytes from 42 healthy volunteers were also assessed as a comparison group. Results We found that CLL samples could be clearly stratified by ZAP-70 expression with a distribution having a peak-trough-peak configuration (Figure). In this way, a definitive threshold was obtained by using the trough as the cut-off. Interestingly, ZAP-70 expression in B cells from healthy persons was unimodal and it peaked in the trough of the distribution of the CLL samples (Figure). Using the ZAP-70 expression trough as a cutoff, unlike the E2997 study, we could delineate patient populations with distinct progression-free survival (p < 0.001) and overall survival (p = 0.004). Moreover, by multivariate analysis ZAP-70 expression was found to be an independent variable (p = 0.01) relating to progression-free survival along with 17p deletion and Rai staging. By assessing the expression levels of the other 28 analytes stratified by ZAP-70 expression, we found statistically significant differences in 10 molecules which included phosphoantigens representing activation levels of various signaling pathways and apoptotic pathway constituents. These findings suggest that ZAP-70 stratification defines 2 distinct types of CLL cells. Besides expression, we found highly significant levels of bivariate correlations between expression levels of the molecules examined including over 30 with correlation coefficients of 0.85 or greater. Interestingly, the correlations varied according to ZAP-70 expression. In samples with high levels of ZAP-70 expression, ZAP-70 was correlated with 13 other analytes with r > 0.5 whereas phospho-ZAP-70 was only correlated with 1 other analyte with the 0.5 threshold. Conversely, in samples with low levels of ZAP-70, phospho-ZAP-70 was correlated with 12 other molecules with r > 0.5 whereas ZAP-70 was correlated with no other analytes. An example showing that ZAP-70 was strongly correlated with Mcl-1 in samples with high levels of ZAP-70 but not in samples with low levels is presented below in the figure. The differential intermolecular relationships seen by ZAP-70 stratification support the possibility that ZAP-70 expression defines 2 different types of CLL cells with clinical outcome implications. Conclusions Our study has uncovered highly significant differential expression levels, intermolecular associations, and organization in CLL samples stratified by ZAP-70 expression. Since stratification by ZAP-70 gave significant prognostic information, we propose that our flow cytometric amplification technology permits characterization of the distinct molecular constitution of the 2 types of CLL cells and provides us with a unique opportunity to understand how the composition of leukemic cells translates into clinical outcome. Additionally, these data provide clues for the development of novel therapies. Disclosures: Kaplan: Pathfinder Biotech: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding. Kaye:Pathfinder Biotech: Employment.

Background and Aim: Serum cancer antigen (CA) 125 elevation has been reported in patients with liver disease, but it is poorly characterized. The present study aimed to evaluate the range of serum and ascitic CA 125 levels in patients with liver cirrhosis and to explore possible factors associated with CA 125 elevation. Methods: A total of 70 patients were studied. Group I consisted of 30 patients with liver cirrhosis with or without ascites. Group II consisted of 30 patients with digestive malignant tumors with or without ascites. Group III consisted of 10 patients with benign ascites. The CA 125 levels were measured in the serum of all patients and also simultaneously in the ascitic fluid of 15 patients. Results: Serum CA125 levels in 80% of (24/30) patients from group I were elevated, particularly in those with ascites, irrespective of the etiology of cirrhosis. Serum CA 125 levels were correlated with Child–Pugh scores (r = 0.38), but not significantly (P = 0.06). All patients from group II with ascites and from group III had elevated serum CA 125 levels, but there was no difference in the serum CA 125 levels between patients with ascites from group I (275 ± 175 U/mL), group II (368 ± 190 U/mL) or group III (396 ± 287 U/mL), nor was there a significant difference in ascitic CA 125 levels (P > 0.05). The levels of serum CA 125 (198 ± 108 U/mL) were lower than, but correlated with that of ascites (460 ± 234 U/mL, r = 0.58, P = 0.026). The elevation of serum CA 125 accompanied by abnormalities of other tumor markers was more common in malignant ascites than in benign ascites (90% compared with 6%, P

AIM: To summarize the characteristics of patients suffered from primary biliary cirrhosis associated with ulcerative colitis. METHODS: To report a new case and review the literature. RESULTS: There were 18 cases (including our case) of primary biliary cirrhosis complicated with ulcerative colitis reported in the literature. Compared with classical primary biliary cirrhosis, the patients were more often males and younger similar. The bowel lesions were usually mild with proctitis predominated. While ulcerative colitis was diagnosed before primary biliary cirrhosis in 13 cases, the presentation of primary biliary cirrhosis was earlier than that of ulcerative colitis in our new case reported here. The prevalence of primary biliary cirrhosis among patients of ulcerative colitis was almost 30 times higher than in general population. CONCLUSION: Association of primary biliary cirrhosis with ulcerative colitis is rare. It should be considered in the differential diagnosis of hepatobiliary disease in patients with ulcerative colitis, and vice versa.

AIM: To determine the changes of CD8+ T subsets especially CD8+CD28- T regulatory cells in rat model of experimental colitis induced by 2,4-dinitrofluorobenzene (DNFB). METHODS: The rat model of experimental colitis was induced by enema with DNFB. Ten days later, colonic intraepithelial and splenic lymphocytes were isolated from colitis animals (n = 16) and controls (n = 8). The proportion of CD8+ T cells, CD8+CD28+ T cells and CD8+CD28- T regulatory cells were determined by flow cytometry. RESULTS: The model of experimental colitis was successfully established by DNFB that was demonstrated by bloody diarrhea, weight loss and colonic histopathology. The proportion of CD8+ T cells in either splenic or colonic intraepithelial lymphocytes was not significantly different between colitis animals and controls (spleen: 34.6% ± 7.24% vs 33.5% ± 9.41%, colon: 14.0% ± 8.93% vs 18.0% ± 4.06%, P > 0.05). But CD8+CD28- T regulatory cells from colitis animals were significantly more than those from controls (spleen: 11.3% ± 2.26% vs 5.64% ± 1.01%, colon: 6.50% ± 5.37% vs 1.07% ± 0.65%, P < 0.05). In contrast, CD8+CD28+ T cells from colitis animals were less than those from controls (spleen: 23.3% ± 6.14% vs 27.8% ± 9.70%, P = 0.06; colon: 7.52% ± 4.18% vs 16.9% ± 4.07%, P < 0.05). The proportion of CD8+CD28- T regulatory cells in splenic and colon intraepithelial CD8+ T cells from colitis animals was higher than that from controls (spleen: 33.3% ± 5.49% vs 18.4% ± 7.26%, colon: 46.0% ± 14.3% vs 6.10% ± 3.72%, P < 0.005). CONCLUSION: Experimental colitis of rats can be induced by DNFB with simplicity and good reproducibility. The proportion of CD8+CD28- T regulatory cells in rats with experimental colitis is increased, which may be associated with the pathogenesis of colitis.