ALBERT

Description: T(11;18)(q21;q21) is the most common structural abnormality in extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) leading to the fusion of the apoptosis inhibitor-2 (API2) gene and the MALT lymphoma-associated translocation (MALT1) gene. In 2 patients with MALT lymphoma of the liver and skin, respectively, t(14;18)(q32;q21) was observed by cytogenetic analysis. Subsequent fluorescence in situ hybridization (FISH) studies disclosed that the immunoglobulin heavy-chain locus (IGH) and the MALT1 gene were rearranged by this translocation. In order to screen a large series of MALT lymphomas for this aberration, a 2-color interphase FISH assay was established. Among a total of 66 cases, t(14;18)(q32;q21) involving IGH and MALT1 was detected in MALT lymphomas of the liver (4 of 4), skin (3 of 11), ocular adnexa (3 of 8), and salivary gland (2 of 11), but did not occur in MALT lymphomas of the stomach (n = 10), intestine (n = 9), lung (n = 7), thyroid (n = 4), or breast (n = 2). In total, 12 of 66 (18%) MALT lymphomas harbored t(14;18)(q32;q21); 7 additional cases of splenic marginal zone lymphoma tested negative. All of the 12 MALT lymphomas featuring the t(14;18)(q32;q21) were negative for t(11;18)(q21;q21) by reverse transcriptase–polymerase chain reaction (RT-PCR). However, trisomy 3 and/or 18 was found in 4 of 12 cases, suggesting that the t(14;18)(q32;q21) does not occur as the sole genetic abnormality. This study identifies IGH as a new translocation partner of MALT1 in MALT lymphomas, which tend to arise frequently at sites other than the gastrointestinal tract and lung. In contrast to t(11;18)(q21;q21)+ MALT lymphomas, those with t(14;18)(q32;q21) may harbor additional genetic abnormalities.

Description: The growth of most tumors depends on the formation of new blood vessels. In contrast to genetically unstable tumor cells, endothelial cells of tumor vessels are believed to be normal diploid cells that do not acquire mutations. We have recently observed that microvessel endothelial cells of patients with B-cell lymphomas carry lymphoma specific aberrations (NEJM351:250–9, 2004). The aim of this study was to determine whether hematological malignancies other than B-cell lymphomas also carry disease specific genetic aberrations. Using a combined immunohistochemical and fluorescence in situ hybridization assay, we investigated the endothelial cells of 5 multiple myelomas, 6 anaplastic large cell lymphomas, 1 angioimmunoblastic T-cell lymphoma, 4 chronic myeloid leukemias, 3 acute myeloid leukemias and 1 acute lymphoblastic leukemia for cytogenetic alterations that were known to be present in the malignant hematopoietic cells. We found that microvascular endothelial cells of all investigated cases harbored the disease-specific chromosomal aberrations. The percentages of genetically aberrant endothelial cells were highest in chronic myeloid leukemia (median 63%) and multiple myeloma (median 42%) and lowest in anaplastic large cell lymphoma (median 15%). Our findings suggest that the genetic relationship between tumor cells and their endothelium is a common phenomenon of hematological malignancies and reflect a novel aspect of tumor angiogenesis.

Description: Abstract 2433 Chronic lymphocytic leukemia (CLL) is characterized by the clonal expansion of B lymphocytes which typically express CD19 and CD5. The disease remains incurable and recurrence often occurs after current standard therapies due to residual disease or probably due to the presence of therapy-resistant CLL precursors. Based on the growing evidence for the existence of leukemia stem cells, this study was designed to search for putative CLL precursors/stem cells based on the co-expression of CLL cell markers (CD19/CD5) with the hematopoietic stem cell marker (CD34). Forty seven CLL patients and 17 healthy persons were enrolled in the study. Twenty four patients had no previous treatment and 23 had pre-therapy. Twenty two patients were in Binet stage C and 25 patients in B. Twenty two patients had unmutated and 18 mutated IgVH gene (7: ND). Cytogenetic analysis by FISH showed that 14 patients had del 13q, 8 had del 11q, 4 had del 17p and 9 had trisomy 12. Peripheral blood and bone marrow mononuclear cells were subjected to multi-colour FACS analysis using anti-human antibodies against CD34, CD19 and CD5 surface antigens. The results revealed the presence of triple positive CD34+/CD19+/CD5+ cells in CLL samples (mean 0.13%; range 0.01–0.41) and in healthy donors (0.31%; range 0.02–0.6) within the CD19+ B cells. However, due to the high leukocyte count in CLL patients, the absolute number of these cells was significantly higher in CLL samples (mean: 78.7; range 2.5–295 cells /μL blood) compared to healthy persons (mean: 0.45: range 0.04–2.5 cells/μl)(p

Description: Abstract 3382 Siglec-3 (CD33) is an established therapeutic target in acute myeloid leukemia (AML). We and others have shown that CD33 is expressed on immature CD34+/CD38- stem cells in AML. We here report that leukemic stem cells obtained from patients with chronic myeloid leukemia (CML) display high levels of CD33, and that CD33 may serve as a potential target in CML. As assessed by multi-color flow cytometry, CD34+/CD38-/CD123+ CML progenitor cells in chronic phase (CP) were found to express significantly higher levels of CD33 compared to normal CD34+/CD38- bone marrow stem cells (figure). By contrast, similar levels of the cell surface antigen MDR1 (CD243) were detected when comparing normal and CML progenitors. In chronic phase (CP) CML, CD33 was found to be expressed homogeneously on most or all CD34+/CD38- stem cells. In patients with accelerated (AP) or myeloid blast phase (BP), CML stem cells also co-expressed CD33, but the levels of CD33 varied from donor to donor, and in one patient, most CML stem cells appeared to be CD33-negative cells. In two patients with CML, CD34+/CD38- cells were highly enriched by cell sorting (purity 〉98%) and found to contain CD33 mRNA in qPCR analysis. The presence of BCR/ABL and thus the leukemic origin of these cells was confirmed by FISH analysis and PCR. We then examined the effects of the CD33-targeted drug gemtuzumab/ozogamicin (GO) on growth of primary CML cells. As assessed by 3H-thymidine uptake, GO produced growth inhibition in leukemic cells in all patients tested (CP, n=13; AP, n=3). The effects of GO on leukemic cell growth were dose-dependent and occurred at relatively low concentrations, with IC50 values ranging between 1 and 100 ng/ml. GO effects were also seen in precursor-enriched Lin-negative CML cells (n=3). As assessed by Annexin-V staining, GO was found to induce apoptosis in CD34+/CD38- CML progenitor cells. Next we investigated drug combination effects. In these experiments, GO was found to synergize with nilotinib and bosutinib in producing growth inhibition in primary CML cells. In conclusion, CD33 is expressed abundantly on immature CD34+/CD38- progenitor cells in CML. Whether GO can be employed to eradicate residual leukemic stem cells in CML patients alone or in combination with BCR/ABL kinase inhibitors remains at present unknown. Figure: Expression of CD33 on CD34+/CD38- cells in normal bone marrow (n=7) and patients with CML in chronic phase (CP, n=16) or advanced phase (AP/BP, n=11) of the disease. Figure:. Expression of CD33 on CD34+/CD38- cells in normal bone marrow (n=7) and patients with CML in chronic phase (CP, n=16) or advanced phase (AP/BP, n=11) of the disease. Disclosures: Valent: Novartis: Research Funding; Bristol-Myers Squibb: Research Funding.

Description: Key Points DPPIV (CD26) is a new specific marker of CML LSC that aids CML diagnostics and the measurement, characterization, and purification of LSC. DPPIV on CML LSC degrades SDF-1 and thereby promotes the niche-escape of LSC, which may contribute to extramedullary myeloproliferation in CML.

Description: In Eμ-myc transgenic animals lymphoma formation requires additional genetic alterations, which frequently comprise loss of p53 or overexpression of BCL-2. We describe that the nature of the “second hit” affects the ability of the immune system to contain lymphoma development. Tumors with disrupted p53 signaling killed the host more rapidly than BCL-2 overexpressing ones. Relaxing immunologic control, using Tyk2−/− mice or by Ab-mediated depletion of CD8+ T or natural killer (NK) cells accelerated formation of BCL-2–overexpressing lymphomas but not of those lacking p53. Most strikingly, enforced expression of BCL-2 prolonged disease latency in the absence of p53, whereas blocking p53 function in BCL-2–overexpressing tumors failed to accelerate disease. This shows that blocking apoptosis in p53-deficient cells by enforcing BCL-2 expression can mitigate disease progression increasing the “immunologic visibility.” In vitro cytotoxicity assays confirmed that high expression of BCL-2 protein facilitates NK and T cell–mediated killing. Moreover, we found that high BCL-2 expression is accompanied by significantly increased levels of the NKG2D ligand MULT1, which may account for the enhanced killing. Our findings provide first evidence that the nature of the second hit affects tumor immunosurveillance in c-MYC–driven lymphomas and define a potential shortcoming of antitumor therapies targeting BCL-2.

Description: Abstract 2685 Genetic alterations such as C-MYC, BCL2, BCL6 translocations and TP53 alterations have a prognostic impact in patients with aggressive B-cell lymphomas (Johnson et al JCO 2012; Sietse et al Blood 2011; Young et al Blood 2009). Here we provide novel information about the influence on survival of sole C-MYC translocation compared with additional BCL2 and/or TP53 alterations. The clinical outcome of 28 patients with C-MYC positive diffuse large B-cell lymphoma (DLBCL) (N=4) or B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and Burkitt lymphoma (B-UNC/DLBCL/BL) (N=24) was retrospectively evaluated according to their genetic alterations (GA) and therapy (R-CHOP N=10 (36%) patients vs. B-ALL-GMALL containing HD-MTX, N=18 (64%) patients). C-MYC, BCL2 and TP53 alterations were screened by FISH. TP53 mutations were investigated by immunohistochemistry, positive cases were further sequenced. Cases were grouped in 4 categories: C-MYC only (32%), C-MYC & BCL2 (32%), C-MYC & TP53 (21%) and triple hit (14%). Statistical analyses were performed using R for Windows 2.12.1 (R Development Core Team) software. P-values 〈 0.05 were considered as significant. Using Kaplan Meier survival analysis no significant difference was found between therapeutic regiments indicating no advantage for aggressive chemotherapy (Fig. 1A). Based on the GA overall survival (OS) was significantly different between the four groups (p=0.048). OS at five years was 75% for C-MYC, 71% for triple hit, 35% for C-MYC & BCL2 and 17% for C-MYC & TP53 (Fig. 1B). These data confirm previous results in MYC-induced murine lymphomas (Schuster et al Blood 2011). In the log-rank test only low International Prognostic Index (IPI) scores (0–1) predicted survival correctly. Due to this fact, we aimed to define a new prognostic index containing genetic parameters. Univariate and multivariate analyses were performed to identify predictive parameters. Patient characteristics were tested for potential influence on OS using the Cox proportional hazards model in combination with all-possible-subset regression. Covariates were: age, LDH (U/ml), BM infiltration (%) and factor variables: sex, LDH high vs. low, presence or absence of BM infiltration, localization (extranodal vs. nodal), clinical stage, IPI-score, therapy and GA. Model selection was performed using the all-possible subset approach, calculating all models with j=1 to 11 covariates. A model was considered valid if all predictors exhibit p-values below 0.05. A factor variable was considered significant if at least one level differs significantly from a predefined base level (base level for GA: sole C-MYC; therapy: GMALL-ALL protocol; stage: CS I; IPI: 0 points). The final model was checked for possible evidence of non-proportional hazards and for influential observations. Table 1. shows the characteristics of the multivariate Cox regression model including the covariates BM and GA (explained variation R2=0.48). Table 1. Model characteristics for the two-variable model containing genetic alterations and BM infiltration as significant covariates. Covariate/factor level Regression coefficient βj Relative Risk exp(βj) p-value 95 % Confidence Interval C-MYC & BCL2 2.8 16.7 0.008 [2.1; 135] C-MYC & TP53 4.3 71.4 〈 0.001 [5.6; 904] C-MYC & BCL2 & TP53 0.2 0.8 0.87 [0.07; 9.1] BM 3.2 25.0 0.001 [3.5; 178] It became obvious that the presence of C-MYC & BCL2 or C-MYC & TP53 increased the relative risk for lymphoma related death whereas C-MYC only and interestingly also triple hit cases showed favorable prognosis in this rare entity. These finding motivated the definition of a new prognostic index: assigning one point each to the risk factors 1) BM infiltration, 2) C-MYC & BCL2; two points to 3) C-MYC & TP53 and no point to 4) presence of sole C-MYC or 5) triple hit aberrations, respectively, yielding scores between 0 and 3. Based on these scores two prognostic groups “good” (scores 0–1) vs. “poor” (score 2–3) were classified. OS stratified by the new risk groups was highly significant (p-value 〈 0.001) (Fig. 1C). Our data indicate that 1) C-MYC and TP53 simultaneous alterations constitute the highest risk group. This risk is considerably ameliorated when an additional BCL2 aberration is present. 2) GMALL-ALL protocol is non-superior to R-CHOP. 3) Our prognostic index integrating clinical and genetic features is able to predict outcome and should be evaluated in larger studies. Disclosures: Jaeger: Emergent Product Development: Consultancy, Research Funding.

Description: Translocations t(11;18)/API2-MALT1, t(14;18)/IGH-MALT1, and t(1;14)/IGH-BCL10 are known to occur in MALT lymphoma and affect a common signalling mechanism, resulting in the constitutive activation of the NF-kB pathway. However, genetic rearrangements are unknown in the majority of MALT lymphoma. In the current study, we investigated the karyotypes of 28 MALT lymphoma of various anatomical sites. Conventional cytogenetics and FISH identified aberrations in 26/28 MALT lymphoma. Balanced translocations were found in 21 cases. IGH was rearranged in the majority of cases with balanced translocations (n=20/21); 1/21 case had novel t(6;7)(q25;q11). IGH partner genes involved MALT1, FOXP1, BCL6 and 4 new chromosomal regions on chromosome arms 1p, 1q, 5q, and 9p. Long-distance inverse polymerase chain reaction identified three novel partner genes on 1p (CNN3), 5q (ODZ2), and 9p (JMJD2C). FISH assays were established and confirmed LDI-PCR results. Real-time quantitative RT-PCR showed up-regulation of the novel genes in the translocation-positive cases. Immunohistochemistry for MALT1, BCL10, FOXP1, and NF-kB was performed in all cases and demonstrated that NF-kB and FOXP1 were not activated in the majority of the cases. Our study shows that MALT lymphoma are genetically heterogenous (at least 10 different translocations) and do not share NF-kB as unifying pathway.

Description: Chronic myeloid leukemia (CML) is a stem cell neoplasm characterized by the Philadelphia (Ph) chromosome and the related oncoprotein, BCR/ABL. Despite the availability of novel BCR/ABL-targeting tyrosine kinase inhibitors (TKI), many patients relapse which may be due to resistance of neoplastic stem cells (NSC) against TKI. So far, little is known about specific markers and targets expressed on CML NSC. We here report that CML NSC express the interleukin-2R alpha-chain CD25 in a specific (aberrant) manner. Whereas normal bone marrow (BM) stem cells were found to express only low amounts or did not express CD25, CD34+/CD38− NSC in CML were found to express CD25 in almost all patients examined (31/33=95%), independent of the phase of disease. CML NSC were also found to express IL-1RAP, DPPIV (CD26), Siglec-3 (CD33), Campath-1 (CD52), KIT (CD117) and IL-3RA (CD123). We were also able to show that highly purified CD25+ NSC express BCR/ABL and engraft irradiated NOD-SCID-IL-2Rg-/- (NSG) mice with BCR/ABL+ cells, whereas CD34+/CD38-/CD25-/CD26- cells from the same patients failed to express BCR/ABL, and engrafted NSG mice with BCR/ABL-negative cells. To define signalling-molecules contributing to the expression of CD25 in CML NSC, we employed primary murine hematopoietic cells infected with bcr/abl-p210 in combination with a retrovirus encoding for STAT5A or STAT5B. Enforced expression of either STAT5A or STAT5B resulted in enhanced expression of CD25 in Lin-/Sca-1+/Kit+ mouse BM stem cells (LSK cells), regardless of the presence or absence of bcr/abl. In fact, the CD25-promoting effect of STAT5 was seen in normal stem cells as well as in bcr/abl-transformed LSK cells. Correspondingly, shRNA against STAT5 was found to downregulate expression of CD25 in the human CML cell line KU812. The BCR/ABL-targeting TKI imatinib, nilotinib and ponatinib were also found to inhibit expression of CD25 on KU812 cells. In primary CML NSC, ponatinib was found to induce marked suppression of CD25, whereas imatinib and nilotinib showed only weak effects. In consecutive experiments, we found that the MEK inhibitors RDEA119 and PD032509, the STAT5-targeting drug pimozide and the dual PI3 kinase/mTOR blocker BEZ235 all inhibit 3H-thymidine uptake and thus proliferation in KU812 cells. However, whereas RDEA119 and PD032509 as well as pimozide were found to downregulate expression of CD25 in KU812 cells, BEZ235 was found to even upregulate expression of CD25 on KU812 cells. We next applied an shRNA against CD25. The shRNA-induced knock-down of CD25 in KU812 resulted in an increased proliferative capacity, suggesting that CD25 may act as a potential tumor suppressor in CML stem- and progenitor cells. Together, our data show that CD25 is a novel functionally relevant marker-antigen and potential tumor suppressor of CML NSC. Expression of CD25 in CML cells appears to be triggered by MEK and STAT5 activity, and may be negatively regulated by the PI3 kinase/mTOR pathway. Whether modulation of CD25 can be exploited clinically and used as basis for the development of novel NSC-targeting treatment concepts in CML remains to be determined in future studies. Disclosures: Valent: Novartis: Consultancy, Honoraria, Research Funding.

Description: Nodal peripheral T-cell lymphomas represent a heterogeneous category, composed of three entities: anaplastic large cell lymphomas, peripheral T-cell lymphomas unspecified (PTCLu) and angioimmunoblastic T-cell lymphomas (AITL). The later entity has been recently recognized to derive from follicular helper T cells (TFH). Among PTCLu - which represents an ill-defined entity - a peculiar form with follicular growth pattern (PTCL-F) has been recently reported, and one article stated their association with t(5 ;9)(q33 ;q22) involving ITK and SYK (Leukemia2006; 20: 313–318). However, the origin of tumor cells and clinical aspects of this group of PTCL-F are still unknown. The aim of this study was to analyse a series of PTCL-F to describe their clinical and histopathological aspects, to identify their cell of origin, and their relationship with AITL. Fourty-two patients from 32 to 85 years of age with 51 biopsies were selected from three Departments of Pathology (Creteil, n=24, Nantes n=13, Vienna n=14). All patients showed histopathologic features of PTCL-F in at least one biopsy. Biopsies were classified into three categories according to predominant morphological features at low power magnification: follicular lymphoma-like (n=7), progressive transformation of germinal center-like (n=22), and AITL-like features with follicular colonization (n=19). Several cases have combinations of patterns. The neoplastic population is characterized by medium-sized cells with clear cytoplasm surrounded by IgD+ B-cells. Tumor cells are of helper T-cell immunophenotype [CD2+ (33/33 = 100%), CD3+ (45/48 = 93%), CD4+ (35/42 = 83%), CD5+ (39/39 = 100%), CD7+ (7/37 = 19%)], with frequent expression of CD10 (29/43 = 67%) and of TFH markers [PDCD-1 (32/36 = 88%), CXCL13+ (33/38 = 87%), BCL6+ (15/25 = 60%), CD57+ (9/16 = 56%)]. Scattered CD20+ B-immunoblasts (27/28 = 96%) and EBV+ cells (18/30 = 60%) are also frequently observed. Seven out of 31 patients (22%) in the 3 morphological patterns have t(5 ;9)(q33 ;q22) detected by fluorescent in situ hybridization. At prentation and/or at relapse, most patients had multiple lymphadenopathies (19/23 = 83%) and disseminated disease (stages III–IV, 22/28 = 79%). Skin lesions and B symptoms were present in 7/19 (37%) and 6/22 (27%) patients, respectively. In addition, 2 patients with sequential biopsies disclosed typical clinical & histopathological features of AITL in one episode. Our results show that this rare form of PTCL has an immunophenotype indicative of TFH origin, is associated with t(5 ;9) in a proportion of cases, shows some similarities in morphology and immunophenotype with AITL, suggesting a relationship, and generates diagnostic pitfalls, especially with atypical reactive lymphoid lesions and some B-cell lymphomas. The use of immunohistochemistry with TFH markers and molecular studies can help to make a correct diagnosis.