Absence Of Bcl-2 Research Articles

Early-Stage Follicular Lymphoma: Learnings from the Final Analysis of the Multicenter Phase II FIL (Fondazione Italiana Linfomi) “Miro” Trial, Combining Local Radiotherapy and MRD-Driven Immunotherapy

Introduction. Early-stage follicular lymphoma (FL) is usually managed with involved field radiotherapy (IFRT), allowing a complete and long-lasting eradication of the disease in 40-50% of patients (pts). The aim of this multicenter phase II prospective study was to evaluate if a minimal residual disease (MRD)-driven immunotherapy consolidation after IFRT can improve the outcome of pts unlikely to be cured by IFRT only, and to assess the therapeutic effect of IFRT and anti-CD20 consolidation by MRD evaluation. Methods. One hundred and ten pts with stage I/II FL were enrolled; 106 were evaluable and treated with 24 Gy IFRT. Peripheral blood (PB) and bone marrow (BM) samples were centralized to the FIL (Fondazione Italiani Linfomi) MRD Network of EuroMRD-certified laboratories. In pts BCL2::IGH+ at baseline by both nested PCR (NEST) and RQ-PCR (RQ) in the BM and/or (a/o) PB, MRD was analyzed after IFRT and every 6 months over a 3-year period. Pts who were MRD+ by both NEST and RQ in the BM a/o PB after IFRT or who became MRD+ during the follow-up were treated with 8 weekly doses of the anti-CD20 monoclonal antibody ofatumumab (OFA). The primary objective of the study was to define the efficacy of immunotherapy in obtaining a negative MRD. Results. Of the 106 evaluable pts, 50 were males (47%). Median age was 55.5 years (29-83). The FLIPI score was 0 in 60% of pts, 1 in 34%, 2 in 6%; 69% of pts had inguinal site involvement, significantly more frequent in young patients. At baseline, 30.5% of pts had a BCL2::IGH rearrangement (30 MBR, 1 MBR and mcr, 1 mcr) in the BM a/o PB; the concordance between compartments was 87%. BCL2::IGH+ and BCL2::IGH- pts were not significantly different, except for a younger age (p 0.009) and a more frequent FLIPI 0 (p 0.052) among marker negative cases. All but 1 pt achieved a clinical response after IFRT; 1 pt died soon after IFRT of unrelated causes. MRD evaluation after IFRT revealed the persistence of BCL2::IGH+ cells in the PB a/o BM in 60% of pts. MRD+ pts, either after IFRT (n=18) or in case of conversion to MRD+ during the follow-up (n=8), received OFA, obtaining a conversion to MRD- in 23/25 evaluable pts (92%, CI 74-99%), significantly superior to the expected 50%. After a median follow-up of 46.3 months (m) (range 11-66), of the 23 pts who achieved a MRD- with OFA, 5 returned MRD+ (4 underwent OFA re-treatment, achieving a second MRD-, 1 relapsed as a transformation), 4 relapsed being MRD-, 14 remain in complete remission and MRD-, with a duration of molecular response at the last molecular assessment of 30 months (range 6-36). Overall, a clinical relapse was observed in 29 pts submitted to IFRT and 1 patient progressed soon after IFRT. A confirmed histologic transformation to DLBCL was observed in 5 of these patients. Among the 73 “marker negative” patients, 20 relapsed (27.4%) over time, whilst among the 32 BCL2::IGH+ at baseline 9 (28.1%) were resistant or relapsed (p=0.939). The administration of OFA was associated to a significantly reduced risk of POD24 (p= 0.035). Overall, the 24-m and 36-m overall survival (OS) were 100% and 98.9%, respectively; the 24-m and 36-m PFS were 83.7% and 77.4%, respectively. PFS was not different according to the presence or absence of BCL2::IGH at enrollment (p=0.820). PFS was significantly different according to the FLIPI score (p=0.041), but not different according to gender (p=0.696), stage I-II (p=0.122), histological grade (p=0.212), inguinal nodal site vs others (p=0.492). The Kaplan Meier landmark analysis, stratified by MRD, at m+6 (p=0.228), m+12 (p=0.003), m+18 (p=0.0002), m+24 (p=0.004) showed that PFS significantly improved from m+12 onwards when a negative MRD was present (Figure 1). Conclusions. IFRT alone is often insufficient to eradicate FL, inducing a MRD- status only in 40% of pts, long-lasting only in a third of them. An immunotherapy consolidation with OFA after IFRT in MRD+ pts was capable of increasing the molecular responses to 92%, allowing to achieve the primary objective of the study, though this effect was not always persistent. The achievement of a molecular remission correlated significantly with a reduction in the incidence of relapse over time, although this strategy was applicable only to 30% BCL2::IGH+ PET/CT staged FL pts. A consolidation strategy with more effective agent should be advisable. With these limits, a clinical advantage of the MRD-driven treatment strategy is suggested, although not largely applicable.

BDA-366, a putative Bcl-2 BH4 domain antagonist, induces apoptosis independently of Bcl-2 in a variety of cancer cell models

Several cancer cell types, including chronic lymphocytic leukemia (CLL) and diffuse large B-cell lymphoma (DLBCL) upregulate antiapoptotic Bcl-2 to cope with oncogenic stress. BH3 mimetics targeting Bcl-2’s hydrophobic cleft have been developed, including venetoclax as a promising anticancer precision medicine for treating CLL patients. Recently, BDA-366 was identified as a small molecule BH4-domain antagonist that could kill lung cancer and multiple myeloma cells. BDA-366 was proposed to switch Bcl-2 from an antiapoptotic into a proapoptotic protein, thereby activating Bax and inducing apoptosis. Here, we scrutinized the therapeutic potential and mechanism of action of BDA-366 in CLL and DLBCL. Although BDA-366 displayed selective toxicity against both cell types, the BDA-366-induced cell death did not correlate with Bcl-2-protein levels and also occurred in the absence of Bcl-2. Moreover, although BDA-366 provoked Bax activation, it did neither directly activate Bax nor switch Bcl-2 into a Bax-activating protein in in vitro Bax/liposome assays. Instead, in primary CLL cells and DLBCL cell lines, BDA-366 inhibited the activity of the PI3K/AKT pathway, resulted in Bcl-2 dephosphorylation and reduced Mcl-1-protein levels without affecting the levels of Bcl-2 or Bcl-xL. Hence, our work challenges the current view that BDA-366 is a BH4-domain antagonist of Bcl-2 that turns Bcl-2 into a pro-apoptotic protein. Rather, our results indicate that other mechanisms beyond switching Bcl-2 conformation underlie BDA-366’s cell-death properties that may implicate Mcl-1 downregulation and/or Bcl-2 dephosphorylation.

Apoptotic Blocks in Primary Non-Hodgkin B-Cell Lymphomas Identified By BH3 Profiling

Background: The anti-apoptotic protein BCL2 is expressed in most non-Hodgkin lymphomas (NHL) including chronic lymphocytic leukemias (CLL), mantle cell lymphomas (MCL), follicular lymphomas (FL), diffuse large B cell lymphomas (DLBCL) and marginal zone lymphomas (MZL). BCL2 expression is associated with an inferior survival when co-expressed with MYC, an oncogene that induces cellular proliferation, especially in high-grade lymphomas with translocations in MYC and BCL2(HGBL-DH). Venetoclax, a selective BCL2 inhibitor, is effective in CLL, but has modest clinical activity in other NHLs. CLL has been shown to be “primed” and BCL2-dependent, predicting for a favorable response to venetoclax using BH3 profiling. This technique measures mitochondrial outer membrane depolarization (MOMP) after exposure to synthetic pro-apoptotic BH3 peptides that have different affinities to anti-apoptotic proteins BCL2, BCLXL, MCL1 and BCLW. Cells with functional BAX/BAK undergo MOMP after exposure to pro-apoptotic proteins BIM/BID. “Primed” cells undergo MOMP after exposure to BIM, BID and PUMA, where the latter inhibits all anti-apoptotic proteins. Thus, inhibiting the anti-apoptotic proteins would initiate apoptosis. We hypothesized that BH3 profiling of NHLs could help us understand the mechanisms of venetoclax-resistance in BCL2+ NHL. This has not yet been performed due to the rarity of samples archived as a viable cell suspension.Methods: We performed BH3 profiling on 138 B-cell NHLs (36 CLL/SLL, 42 FL, 38 DLBCL, 10 HGBL-DH, 13 MCL, and 3 MZLs) and 34 controls (14 tonsils and 20 peripheral blood mononuclear cells). NHLs were taken prior to (n=107) or after therapy (n= 31). A T-test and ANOVA were performed to determine the differences in MOMP between NHLs subtypes and normal B cells. We also profiled HGBL-DH cell lines to determine if chemotherapy (cyclophosphamide, doxorubicin, vincristine, dexamethasone and bendamustine) could potentiate venetoclax-responses. We measured the levels of anti-apoptotic proteins and MYC by immunoblot, speculating that proteins with short half-lives (MYC and MCL1) may fall upon cell-cycle arrest.Results: Normal B and T lymphocytes were primed and depended on BCL2, MCL1 and BCL-XL. NHLs were primed in 85% (121/138) of cases, 5% (6/138) were unprimed (no PUMA response) and 10% (14/138) were incompetent to undergo MOMP (no BIM/BID response), indicating dysfunctional BAX/BAK. The latter apoptotic defect was mainly seen in DLBCL (10/14). CLL and HGBL-DH had significantly higher venetoclax responses than DLBCL, MCL and FL (all p <0.05). Responses to venetoclax were only observed in samples that expressed BCL2 protein. In BCL2+ NHLs, increased MOMP was achieved by combining venetoclax with the MCL1-specific inhibitor MS1. Inhibiting BCLXL or BCLW did not increase MOMP, suggesting that MCL1 inhibits apoptosis in the absence of BCL2. Profiling HGBL-DH cell lines OCI-Ly8 and Toledo after chemotherapy, only doxorubicin and vincristine increased priming (PUMA response), decreased the levels of MYC and MCL1 proteins and potentiated venetoclax-induced apoptosis (all p <0.05).Conclusion: CLL and HGBL-DH were the most BCL2-dependent NHLs and had the highest responses to venetoclax. Venetoclax-resistance in our NHLs was due to the presence of MCL1, the lack of BCL2 expression and dysfunctional BAX/BAK. In the former scenario, resistance may be overcome by combining venetoclax with vincristine and doxorubicin, two drugs used in standard RCHOP. DisclosuresJohnson:Seattle Genetics: Honoraria; Merck: Consultancy, Honoraria; AbbVie Inc.: Consultancy, Honoraria, Research Funding; Lundbeck: Consultancy, Honoraria, Other: travel, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Other: travel, Research Funding. Steidl:Juno Therapeutics: Consultancy; Roche: Consultancy; Bristol-Myers Squibb: Research Funding; Tioma: Research Funding; Seattle Genetics: Consultancy; Nanostring: Patents & Royalties: patent holding. Scott:Janssen: Research Funding; NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies, Research Funding; Celgene: Consultancy, Honoraria; Roche: Research Funding. Letai:Flash Therapeutics: Equity Ownership; Vivid Biosciences: Equity Ownership; AbbVie: Consultancy, Other: Lab research report; Novartis: Consultancy, Other: Lab research report; AstraZeneca: Consultancy, Other: Lab research report.

Pediatric-Type Nodal Follicular Lymphoma in Children and Adults Is Nearly Genetically Silent and Biologically Distinct from Typical Follicular Lymphoma

Pediatric-type nodal follicular lymphoma (PTNFL) is a variant of follicular lymphoma (FL) characterized by localized presentation and excellent behavior despite its often high-grade histologic appearance. We recently identified high proliferation index and the absence of BCL2, BCL6, and IRF4 gene rearrangements as defining features of PTNFL in both children and adults (Louissaint, Blood 2012). In contrast to typical FL, children with PTNFL consistently have persistent remissions after surgical excision alone. Therefore, it is critically important to identify PTNFL in order to avoid unnecessary therapy. Considering the unique clinical behavior of PTNFL, we hypothesized that the mutational landscape of this disease would differ from that of typical FL. Twenty-four cases were collected from several institutions (MGH, Brigham & Women's, Weill Cornell, Chicago Children's, Boston Children's, University of Pittsburgh, SUNY Downstate, and Stanford). PTNFL was defined by the following criteria: 1) nodal involvement, 2) architectural effacement by a clonal follicular proliferation, 3) high proliferation index and 4) absence of both MUM1/IRF4 expression and BCL2/BCL6 rearrangements by FISH. All cases presented with localized nodal involvement and demonstrated no evidence of recurrence or progression after chemotherapy (n=5), radiation (n=3) or surgical excision alone (n=13) (therapeutic approach to be confirmed in 3 cases), with a median follow-up of 33.1 months (range 10-124 months). Subjects ranged in age from 4-60 years, including 14 children (4-18 years; median 15) and 10 adults (20-60 years; median 29.5). Whole exome sequencing performed on 7 PTNFL cases showed a strikingly low mutation burden (7.1 non-silent mutations/exome), more than an order of magnitude lower than the exomic mutation burden of typical FLs (Green, PNAS 2015; Pasqualucci, Cell Rep 2015). Given these findings, we pursued targeted exome capture and next-generation sequencing for 112 genes previously reported to be recurrently mutated in FL across a panel of 20 PTNFLs. Targeted sequencing (mean depth, 250) again demonstrated very low mutation burden in PTNFL, with a median number of non-silent mutations of 1.67/case compared with 4 mutations/case (P<0.001) in our published analysis of ~300 typical FLs requiring therapy usung the same gene panel (Pastore, Lancet Onc 2015). There were no differences in the median frequencies of mutations among adult and pediatric PTNFLs (1.57/case vs 1.80/case; p=0.752). We also sequenced 17 limited-stage typical FLs using the same 112 gene panel. TNFRSF14 was the most frequently mutated gene in PTNFL, with similar frequencies in PTNFL and limited-stage typical FL (29% vs. 41%; p=0.76). CREBBP mutations, which occur in most cases of advanced-stage FL, were seen in 82% of limited-stage typical FLs and only 4% of PTFLs (p<0.0001). Mutations in all other commonly mutated genes (e.g. BCL2, MLL2, GNA13) were also present in <10% of PTNFLs. SNP-based whole-genome analysis using OncoScan FFPE Assay kit to interrogate copy number was performed on 17 cases of PTNFL and 11 cases of limited stage typical FL. PTNFLs showed an average of 0.18 alterations/case compared with 4.27 alterations/case in limited stage FLs (p<0.0001). Taken together, these molecular genetic findings demonstrate that PTNFL in both children and adults has a strikingly low number of genetic aberrations, including an absence of mutations common in typical FL. The findings provide further evidence that PTNFL has a unique biology that is shared in adult and pediatric cases but is distinct from both limited and advanced stage typical FL. The absence of mutations in commonly mutated genes is an additional criterion, along with clinical and histologic factors, that may guide the diagnosis of PTNFL in both children and adults. DisclosuresSouth:Lineagen Corporation: Consultancy; ARUP Laboratories: Employment; Affymetrix: Consultancy, Honoraria; Illumina: Consultancy, Honoraria. Hasserjian:Promedior: Consultancy.

Risk of CNS Relapse with Diffuse Large B-Cell Lymphoma (DLBCL) in the Rituximab Era: Results from the UK NCRI R-CHOP 14 v 21 Trial

Risk of CNS Relapse with Diffuse Large B-Cell Lymphoma (DLBCL) in the Rituximab Era: Results from the UK NCRI R-CHOP 14 v 21 Trial

Abstract 3833: Molecular mechanisms by which BAY 80-6946 induces apoptosis in breast tumor cells as single agent or in combination

Abstract PI3K/PTEN/AKT pathway has been found frequently activated and mutated in human breast cancer, which contributes to the development and progression of breast cancer as well as drug resistance. As genetic alteration of PIK3CA and PTEN as well as PI3K pathway activation are observed in almost all breast cancer categories, it is important to define the strategy for the development of PI3K inhibitors in breast cancer. BAY 80-6946 is a highly potent and selective pan-class I PI3K inhibitor in PhI clinical development. Molecular profiling of BAY 80-6946 in 24 breast tumor cell lines indicated that tumor cells with either PIK3CA mutation and/or HER2 expression were extremely sensitive to BAY 80-6946 with an average IC50 value of 17 (n=7) and 19 nM (n=8), respectively. In contrast wild type PIK3CA and HER2- breast tumor cells (n=12) were relatively insensitive or resistant to BAY 80-6946 with an average IC50 value of 773 nM. With regard to apoptosis induction, BAY 80-6946 demonstrated superior activity against other PI3K pathway inhibitors tested in PIK3CA mutant breast tumor cells. We found that the expression of Bcl-2, but not Mcl-1 nor survivin determined the sensitivity to apoptosis. Tumor cells that lack Bcl-2 expression immediately underwent apoptosis after exposure to BAY 80-6946. On the other hand, cells expressing high level of Bcl-2 such as T47D (PIK3CAmut) were resistant to the apoptosis induced by BAY 80-6946, despite a potent anti-proliferative activity of BAY 80-6946 (single-digit nM IC50). Combining BAY 80-6946 with ABT737 sensitized T47D cells to apoptosis, further supporting our hypothesis. On the other hand, PTEN-null, EGFRhigh and Bcl2 negative MDA-MB-468 tumor cells, are resistant to apoptosis induction by BAY 80-6946. Using shRNA and inhibitor against mTOR (TORC1 or TORC2), we identified an mTOR-dependent, PI3K-independent phosphorylation of AKT at S473, which might cause tumor cells resistance to the PI3K inhibition by BAY 80-6946. Rictor knockdown or combination with rapamycin can sensitize MDA-MB-468 to BAY 80-6946 with the respect to inhibition of proliferation and induction of apoptosis. Interestingly, the dual PI3K/mTOR inhibitor BEZ-235 could not induce apoptosis in MDA-MB-468, suggesting that the ratio of PI3K vs mTOR inhibition might be important in apoptosis induction. In conclusion, BAY 80-6946 was extremely effective to induce apoptosis in breast cancer cells expressing Her2 and/or PIK3CA mutation in the absence of Bcl-2. BAY 80-6946 in combination with anti-Bcl agents or mTOR inhibitors might be the promising approach to achieve tumor responses in Bcl-2 positive tumors or EGFR expressing/PTEN-null breast tumors, respectively. These findings provide a rationale to develop personalized therapies for the treatment of molecular subtypes of breast cancer.

T(8;22)(q24;q11) In a Context of Complex Karyotype In Two Patients with B-Cell Lymphoma with Features Intermediate Between Burkitt Lymphoma and Diffuse Large B Cell Lymphoma B (LB/LDCGB)

AbstractAbstract 5093 Background:In some cases, it is difficult to distinguish between the diagnosis of Burkitt lymphoma (LB) and Diffuse Large Cell Lymphoma B (DLCL-B) with MYC gene rearrangement. The WHO classification distinguishes three clinical entities: LB, LB/DLCL-B, DLCL-B on the basis of morphology, immunophenotype and genetic characteristics of malignant cells. The MYC gene is often rearranged with the gene that encodes the heavy chains of immunoglobulins (IgG 14q32) and, less frequently, with the gene that encodes the light chains kappa (2p12) or lambda (22q11). Objective:Describe clinical and biological characteristics of two cases of lymphoma with variant t(8;22)(q24;q11) in a complex karyotype diagnosed BL/DLCL-B according to the WHO classification. Methods and Patients:The cytogenetic study was made in the bone marrow (aspirate or biopsy). Leukocytes were incubated during 24h in RPMI without mitogens. Rearrangements of C-MYC, BCL-2, BCL-6 and IGH were studied for FISH techniques. The results were expressed according to ISCN 2009. Case 1:A man of 46 years who was admitted with symptoms of spinal cord compression with fever and weight loss. During admission were detected anti-HIV antibodies. In the biopsy were observed large cells with irregular nuclei, with clefts and some multilobulated. Imunohistochemistry: CD10 +, CD20 +, CD79 +, bcl2 + and BCL6 +. The Ki67/MIB1 was 95%. Cytogenetic study of bone cylinder showed a complex karyotype: 49, XY, +1, del (1) (p22), der (2), der (3), +7, t (8;22)(q24;q11), +10, del(10)(q24q26)x2 [3]. The FISH study demonstrated the rearrangement of C-MYC and the absence of BCL2 and BCL6 rearrangement. Case 2:A man of 67 year old was admitted for epigastric pain, nausea, vomiting and weight loss. The aspirated cells were of medium and large size, irregularly shaped nuclei, some creases and other poly-lobed with prominent nucleoli. Was detected by immunohistochemistry positivity: CD10, CD20, CD79 and BCL6 Ki67/MIB1 presented a 90%. Cytogenetic study of the bone marrow showed a complex karyotype: 49, XY, t(1;11)(q21;p13),+7, t(7;13) q32;q12), t (8;22)(q24;q11),+12,+19[40]. The FISH study demonstrated presence of the rearrangement of C-MYC and BCL2 and BCL6 rearrangement absence. Conclusions:- Both cases are representative of the clinical entity BL// DLCL-B recently recognized by the WHO: anisocitosicas cells with a nucleus of irregular or poly-lobed, with Ki67/MIB1 <90% and C-MYC rearranged with a complex karyotype. - Both cases showed the t(8;22)(q24;q11) rare variant of Burkitt lymphoma and a trisomy 7, trisomy usually observed in DLCL-B. Disclosures:No relevant conflicts of interest to declare.

Opposing effects of bim and bcl-2 on lung endothelial cell migration.

Integration of cell adhesive, survival, and proliferative processes is essential for capillary morphogenesis of endothelial cells (EC) in vitro and vascular development and function in vivo. Unfortunately, the molecular and cellular mechanisms that impact these processes are poorly defined. Here we examined how lack of bim and/or bcl-2 expression impact lung EC function. The absence of bcl-2 or bim had a significant impact on EC adhesion and migration. Lack of bcl-2 expression decreased lung EC migration, whereas lack of bim expression increased migration compared with their wild-type counterparts. Decreased adhesion to fibronectin and vitronectin was observed in both bcl-2-/- and bim-/- lung EC, with bcl-2-/- EC having very little adhesion to either matrix protein. Capillary morphogenesis was greatly diminished in bcl-2-/- EC, which correlated with decreased lung alveolarization in vivo, an angiogenesis-dependent process. We also observed aberrant production of extracellular matrix proteins, eNOS expression, and nitric oxide production in bcl-2-/- lung EC, which could contribute to inability to undergo capillary morphogenesis. The changes in cell adhesion and migration noted in the absence of bim or bcl-2 were independent of their impact on apoptosis. We observed no significant affect on the steady-state rate of apoptosis of lung EC in the absence of bim or bcl-2. Thus, bcl-2 family members, bim and bcl-2, play a central role in modulation of EC proangiogenic properties, which goes beyond their role as simple mediators of mitochondrial homeostasis and apoptosis.

Evaluation of ultraviolet light toxicity on cultured retinal pigment epithelial and retinal ganglion cells

Our study is aimed at evaluating the role of UVB light in inducing cytotoxicity in an in vitro model. RGC-5 and ARPE-19 cells were exposed to different time periods of UVB light: 0, 15, 30, and 45 min. They were subsequently examined for changes in cell morphology, cell viability (neutral red uptake assay), generation of reactive oxygen species (ROS), expression of bax, bcl-2 and cytochome C by reverse transcriptase polymerase chain reaction and western blot, respectively. Dose-dependent reduction in cell viability to UVB light was demonstrated with parallel increase in ROS. Increased duration of exposure (>15 minutes), was associated with increased expression of bax and cytochrome C, and absence of bcl-2 expression. UVB light exposure results in cell cytotoxicity. The concomitant generation of ROS and expression of apoptotic markers suggests the role of oxidative stress in UVB-mediated apoptosis in an in vitro model of retinal ganglion and pigment epithelial cells.

Prognostic Value of p53 and bcl-2 Expression in Patients Treated with Breast Conservative Therapy

Prognostic value of p53 and bcl-2 expression on treatment outcome in breast cancer patients has been extensively evaluated, but the results were inconclusive. We evaluated the prognostic significance of these molecular markers in patients treated with breast conserving surgery and radiotherapy. One hundred patients whose immunostaining of p53 and bcl-2 expression was available among 125 patients who underwent radiotherapy after breast conserving surgery and axillary lymph node dissection were enrolled into this study. Eighty-seven patients also received adjuvant chemotherapy and/or hormonal therapy. Conventional clinicopathologic variables and treatment-related factors were also considered. The 5-yr loco-regional relapse-free and distant metastasis-free survival rates were 91.7% and 90.9%, respectively. On univariate analysis, age, T stage and the absence of bcl-2 & estrogen receptor (ER) expression were associated with loco-regional relapse-free survival. When incorporating these variables into Cox proportional hazard model, only bcl-2(-)/ER(-) phenotype was an adverse prognostic factor (P=0.018). As for the distant metastasis-free survival, age, T stage, and p53 expression were significant on univariate analysis. However, p53 expression was the only prognosticator on multivariate analysis (P=0.009). A bcl-2(-)/ER(-) phenotype and p53 expression are useful molecular markers predicting loco-regional relapse-free and distant metastasis-free survival, respectively, in patients treated with breast conserving surgery and radiotherapy.

Burkitt lymphoma versus diffuse large B-cell lymphoma

Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL) represent distinct entities among aggressive B-cell non-Hodgkin lymphomas (B-NHLs) in the WHO classification [1]. According to the clinical setting of the occurrence, endemic, sporadic and immunodeficiency-associated BL can be distinguished as clinical variants. The diagnosis of classical BL rests on the presence of a monotonous infiltrate of mediumsized blastic lymphoid cells that show round nuclei with clumped chromatin and multiple, centrally located nucleoli. The tumor cells have a high proliferation rate and intermingled macrophages containing apoptotic debris lead to the morphological aspect of a ‘starry sky’ [1] pattern. Immunophenotypic features of BL include positivity of tumor cells for CD20 and CD10 (and BCL6), negativity for BCL2 and a proliferation fraction measured by Ki-67 immunohistochemistry of nearly 100%. In addition, a chromosomal rearrangement of MYC, usually in form of the classical translocation t(8;14)(q24;q32) should be demonstrated at the molecular level. In contrast, DLBCL is characterized by the proliferation of large neoplastic B-cells comprising centroblastic, immunoblastic, T-cell/histiocyte-rich and anaplastic morphological variants [1]. While the differential diagnosis between BL and DLBCL may appear clear-cut in theory, daily practice shows that aggressive B-NHLs are encountered that display some (but not all) morphologic, immunophenotypic and genetic features of classical BL. For these cases, the terms ‘atypical BL’ or ‘Burkitt-like lymphomas’ have been coined. According to the criteria of the WHO, the term ‘atypical BL’ denotes true BL with some unusual—for the most part cytological—features, while the term ‘Burkitt-like lymphoma’ is a non-compromising diagnosis. Hence, whether these cases are biologically and clinically closer to BL or DLBCL is currently a matter of debate. For example, are aggressive B-NHLs with morphological features of classical BL, positivity for CD20 and CD10 and a Ki-67 index of 100% ‘true’ BL, when they show coexpression of BCL2? Likewise, do ‘true’ BL exist that lack genetic rearrangements of MYC? Clinically, the diagnosis of the hematopathologist is of major importance, since treatment regimens for BL [2] and DLBCL [3] differ significantly. Specifically, adult BL are frequently treated with high-intensity chemotherapy regimens including central nervous system prophylaxis leading to an overall survival of 50–70% of these patients [2]. Classical BL, i.e. B-NHL that fulfills all of the above mentioned diagnostic criteria, can be highly reproducibly diagnosed among hematopathologists and, therefore, these cases do not represent a problem—neither with respect to their proper classification nor to the therapeutic implication of the diagnosis. However, what does the diagnosis ‘atypical BL’ or ‘Burkitt-like lymphoma’, which has an unacceptably low interand intra-observer reproducibility [4] imply for the treating hematologist and where should pathologists draw the line between the diagnosis of atypical BL and DLBCL? On the genetic level, translocations involving MYC are a hallmark feature of classical BL. Information on the MYC translocation status, however, is insufficient to discriminate between BL and DLBCL, since 5–10% of DLBCLs also carry a MYC translocation [5]. In recent years, several groups have attempted to establish molecular categories within the gray zone between BL and DLBCL. In a study by Haralambieva and colleagues [6], two independent approaches were tested to assign gray zone lymphomas either to the BL or to the DLBCL category. One approach used only information on morphology, immunohistochemistry, age of the patient and site of involvement (without knowledge of any molecular data), whereas in an independent approach immunohistochemical and molecular markers recommended by the WHO classification (positivity for CD10, BCL6, Ki-67 > 90%, negativity for BCL2; presence of aMYC breakpoint, but absence of BCL2 and/or BCL6 breakpoints) were used as a basis for the distinction. In a control group of pediatric BLs, both algorithms were in agreement in 100% of cases. However, only 20% of the investigated gray zone cases fulfilled the criteria of both algorithms suggesting that only a minority of these cases may represent bona fide BL [6]. Nakamura and colleagues [7] investigated 18 cases of B-NHL with proven MYC rearrangement, which were classified as BL or DLBCL based on histological features. Although BLs were characterized by a significantly higher Ki-67 index than DLBCLs and, vice versa, DLBCLs were more frequently positive for BCL2, this study demonstrated a significant overlap between these two groups. A practical approach to the subdivision of highly proliferative BNHL within the spectrum of BL and DLBCL was recently suggested by Cogliatti and coworkers [8]. Based on available information on morphology (categorized as classical BL, atypical BL and DLBCL), immunophenotype (CD20, CD10, The authors report no relationships with companies whose products or services are mentioned in this article.

Attenuation of retinal endothelial cell migration and capillary morphogenesis in the absence of bcl-2

Apoptosis plays a critical role during development and in the maintenance of the vascular system. B-cell leukemia lymphoma 2 (bcl-2) protects endothelial cells (EC) from apoptosis in response to a variety of stimuli. Previous work from this laboratory demonstrated attenuation of postnatal retinal vascular development and retinal neovascularization during oxygen-induced ischemic retinopathy in bcl-2-deficient (bcl-2-/-) mice. To gain further insight into the function of bcl-2 in the endothelium, we isolated retinal EC from bcl-2+/+ and bcl-2-/- mice. Retinal EC lacking bcl-2 demonstrated reduced cell migration, tenascin-C expression, and adhesion to vitronectin and fibronectin. The bcl-2-/- retinal EC also failed to undergo capillary morphogenesis in Matrigel. In addition, using an ex vivo angiogenesis assay, we observed reduced sprouting from aortic rings grown in culture from bcl-2-/- mice compared with bcl-2+/+ mice. Furthermore, reexpression of bcl-2 was sufficient to restore migration and capillary morphogenesis defects observed in bcl-2-/- retinal EC. Mechanistically, bcl-2-/- cells expressed significantly less endothelial nitric oxide synthase, an important downstream effecter of proangiogenic signaling. This may be attributed to increased oxidative stress in the absence of bcl-2. In fact, incubation of retinal EC or aortic rings from bcl-2-/- mice with the antioxidant N-acetylcysteine rescued their capillary morphogenesis and sprouting defects. Thus, bcl-2-mediated cellular functions play important roles not only in survival but also in proangiogenic phenotype of EC with a significant impact on vascular development and angiogenesis.

Absence of Bcl-2 and Fas/CD95/APO-1 predicts the response to immunotherapy in metastatic renal cell carcinoma

Absence of Bcl-2 and Fas/CD95/APO-1 predicts the response to immunotherapy in metastatic renal cell carcinoma

Lack of BCL-2 confers interferon-alpha sensitivity to B-cell lymphomas

Hairy cell leukemia (HCL) is a chronic B-cell lymphoproliferative disorder with pathological manifestations usually including splenomegaly and panctopenia. Interferons (IFNs), specifically of the α subtypes have shown a significant anti-tumor effect in HCL patients, with improvement of hematological parameters within the first few months of treatment. However, the therapeutic effect of IFN-α is still rather limited. The mechanisms responsible for the beneficial action of IFN-α in HCL patients are unclear. A continuous line of cells (Eskol) from a patient diagnosed with HCL was established and shown to have several properties of HCL. Even though, Eskol cells are very resistant to anti-proliferative activity of IFN-α, Daudi cells, another human B-cell-derived cell line, are very sensitive to anti-proliferative activity of IFN-α and are commonly used as a model cell to test anti-proliferative effect of IFN-α. To understand the molecular reason(s) behind the observed obvious differences to IFN sensitivity of above cells, we have analyzed the expression levels of BCL2, caspase-1, Laminin and PARP in these cells. We found that Daudi cells do not express BCL2 at all, and probably because of that, these cells have constantly cleaved, and probably activated form of caspase-1. However, when we overexpresed BCL2 in these cells, they lost processed form of caspase-1 and became resistant to anti-proliferative activity of IFN-α. These results let us to suggest that IFN-α sensitivity of B-cell lymphomas, once again, depends on the presence or absence of BCL2.

Bcl‐2 expression modulates cell adhesion and migration promoting branching of ureteric bud cells

Bcl-2 is the founding member of a family of proteins that influence apoptosis. During kidney development bcl-2 not only acts as a survival factor, but may also impact cell adhesive mechanisms and by extension branching morphogenesis. The interrelationship between cell adhesion, migration and apoptosis, important during development, is poorly understood. Here we examined the impact lack of bcl-2, an inhibitor of apoptosis, has on ureteric bud (UB) cell adhesion, migration, and branching morphogenesis. Bcl-2 -/- UB cells demonstrated increased cell migration, increased cell invasion and decreased adhesion to vitronectin and fibronectin compared with wild-type cells. Bcl-2 +/+ UB cells readily branched in collagen gel and Matrigel while bcl-2 -/- UB cells did not undergo significant branching in either matrix. Re-expression of bcl-2 in bcl-2 -/- UB cells restored their ability to undergo branching morphogenesis in Matrigel. Consistent with our in vitro data, we show that in the absence of bcl-2, embryonic kidneys undergo decreased UB branching. We observed decreased numbers of UB branch points, UB branch tips and a decreased distance to the first UB branch point in the absence of bcl-2. The alterations in bcl-2 -/- UB cell adhesion and migration was also associated with a significant alteration in expression of a number of extracellular matrix proteins. Bcl-2 -/- UB cells exhibited increased fibronectin expression and decreased thrombospondin-1 and osteopontin expression. Taken together, these data suggest that bcl-2 is required for the proper regulation of cell adhesive and migratory mechanisms, perhaps through modulation of the cellular microenvironment.

Absence of Bcl-2 and Fas/CD95/APO-1 predicts the response to immunotherapy in metastatic renal cell carcinoma

Immunotherapy is the only available treatment for metastatic renal cell cancer (RCC), but the response rate is only about 20% and the treatment is occasionally associated with severe adverse effects. Thus, the selection of patients with a high susceptibility to immunotherapy is needed; however, there is no promising molecular marker that can predict the response to immunotherapy for RCC. This study was carried out to elucidate the potential role of apoptosis-related molecules Bcl-2 and Fas, as well as apoptotic and proliferating indexes (AI, PI) as predictors of the susceptibility of metastatic RCC to immunotherapy. Immunohistochemical examination of tumour tissues from 40 patients with metastatic RCC undergoing postoperative immunotherapy after radical nephrectomy was performed. Patients with progressive disease (PD) after immunotherapy presented with decreased survival (P=0.006). Progressive disease correlated with higher PI in the primary lesion (P=0.0087). All primary tumours of CR or PR patients were negative for Bcl-2, whereas among NC+PD patients, 40.6% were positive for Bcl-2 (P=0.0373). Patients in whom the primary tumours were both Bcl-2- and Fas-negative showed significantly better responses to immunotherapy in comparison with the remaining group (P=0.0022). The Bcl-2 and Fas status of the primary lesion may become useful criteria for the selection of patients with metastatic RCC for immunotherapy.

One single dose of rituximab added to a standard regimen of CHOP in primary treatment of follicular lymphoma appears to result in a high clearance rate from circulating bcl- 2/IgH positive cells: Is the end of molecular monitoring near?

Accurate monitoring of the t(14;18) translocation is regarded as highly desirable in patients with follicular lymphoma (FL) as absence of bcl-2/IgH positive cells has been correlated with a reduced risk of recurrence and, more recently, pre-treatment t(14;18) load with response to rituximab (R; Blood 2004;103:4416-23). With the arrival of R clinical and molecular remission rates for various lymphoma entities improved significantly, creating the need to carefully review and reassess the role of PCR negativity for clinical outcome, specifically when considering the prolonged presence of the drug as compared to chemotherapy. To determine the rate of molecular clearance achieved by the addition of different doses of R 16 newly diagnosed, t(14;18) positive patients with FL (Ann Arbor stages III/IV) were investigated before, during and after primary chemotherapy with six cycles of CHOP combined with varying (1, 3 or 6) cycle numbers of R (varR1-, varR3- or varR6-CHOP, respectively) regarding molecular remission status. For this purpose classic nested PCR as well as a newly established RQ-PCR employing juxtaposed hybridisation probes were employed to assess molecular remission prior, during and post therapy. Interestingly, administering just a single cycle of R (varR1-CHOP) in combination with a standard regimen of CHOP resulted in rapid and effective clearance of t(14;18) carrying cells from the peripheral blood of 4/5 patients in this treatment group. 6/8 (6/8) varR1-/varR3-CHOP patients were fully cleared and 8/8 (7/8) var6-CHOP patients as assessed by RQ- (nested) PCR. This indicates the high clearance capacity of this combination therapy approach even when adding a very low cycle number of R (1 and 3, respectively) to CHOP in primary therapy of FL. In summary, the relationship established between molecular clearance and minimal residual disease/risk of recurrence may have to be redefined in the times of R. Upcoming large prospective trials will have to elucidate to what degree the clearance of peripheral blood from t(14;18) positive cells can still be regarded as informative regarding absence of minimal residual disease, remission status and/or risk of recurrence.

Indispensable role of Bcl2 in the development of the melanocyte stem cell

Bcl2 null mice display a characteristic loss of pigmentation demonstrating the importance of Bcl2 in the melanocyte (Mc) lineage. It was recently reported that this abnormal phenotype is due to the failure of melanocyte stem cell (MSC) maintenance and that Bcl2 is selectively important for the survival of MSCs. However, in our analysis of the same mouse, we observe a reduction in melanoblast (Mb) number in both epidermal and follicular populations. More importantly, there is a complete absence of MSCs. SCF downregulation in the epidermis is concomitant with the dramatic reduction in Mb numbers observed in the Bcl2 null, suggesting that Bcl2 is indispensable for the survival of Mbs in the absence of c-Kit signaling. Consistently, abrogation of c-Kit signaling in Bcl2 null mice depletes all Mbs and Mcs, whereas continuous expression of SCF in epidermal keratinocytes rescues the MSCs. Our results demonstrate that Bcl2 has a general role in Mb and Mc survival and is essential for the emergence of MSCs. Moreover, the results indicate that the first wave of Mcs that provide hair pigmentation is derived directly from epidermal Mbs bypassing MSCs. Furthermore, a Bcl2-independent mechanism of action of SCF in the Mc lineage is revealed as SCF c-Kit signaling is functional in the absence of Bcl2.

Bcl2 Associated Induction of the Suppressor of Cytokine Signaling-3 (SOCS3) Gene Involves Activation of the p44/42 MAPK Pathway.

SOCS3 has been shown to be an essential regulator of IL-6 signaling in hepatocytes and macrophages, as well as a negative regulator of G-CSF signaling in myeloid cells, and SOCS3 expression appears to be STAT3-dependent. However, the underlying cellular pathways which regulate SOCS3 expression in B cells remain to be clearly defined. We previously showed that polyclonal CD19+ B cells isolated from Bcl2 overexpressing Eμ-Bcl2 transgenic mice overexpress SOCS3 protein and that exogenous expression of Bcl2 in hematopoietic and fibroblast cell lines induces SOCS3 protein expression. We also found that a distinct subset of follicular lymphomas (FL) exhibit combined overexpression of Bcl2 and SOCS3. We hypothesized that Bcl2-mediated induction of SOCS3 in B cells may occur indirectly via deregulation of Bcl2-associated cell signaling pathways. In the current study, IL-3-dependent BaF3 pro-B cells were stably transduced with either a 717bp human Bcl2α cDNA (BaF3/Bcl2) or vector only control (Baf3Δ). Cell lines were initially grown in the presence of IL-3, with IL-3 removed 48 hours prior to protein measurements. Western analysis using Bcl2 antisera revealed high level Bcl2 expression in transduced cells (BaF3/Bcl2) and absence of Bcl2 in BaF3Δ control cells. When probed with SOCS3 antisera, BaF3/Bcl2 cells exhibited marked overexpression of SOCS3 protein whereas BaF3Δ control cells did not express SOCS3. To assess whether Bcl2-associated induction of SOCS3 is STAT3-dependent, we measured phospho-STAT3 levels relative to STAT3 in BaF3/Bcl2 cells. Interestingly, when probed with phospho-STAT3 and STAT3 antisera, BaF3/Bcl2 and BaF3Δ cells did not reveal phospho-STAT3 protein but revealed equivalent STAT3 protein levels. Since we had previously noted overexpression of p44/42 Map kinase (MAPK) in CD19+ B cells from Eμ-Bcl2 transgenic mice relative to littermate controls, we next wanted to determine whether p44/42MAPK signaling played a role in Bcl2-mediated SOCS3 induction. Western analysis using p44/42MAPK antisera revealed overexpression of p44/42MAPK in BaF3/Bcl2 cells relative to BaF3Δ control cells. BaF3/Bcl2 and BaF3Δ cells were then grown in the presence or absence of a specific p44/42MAPK pathway inhibitor (MEK 1/2 inhibitor U0126), and whole cell lysates were analyzed for SOCS3 expression by Western analysis. When probed with SOCS3 antisera, BaF3/Bcl2 cells selectively grown in the presence of the p44/42MAPK inhibitor revealed complete abrogation of SOCS3 protein expression while those grown in the absence of inhibitor continued to harbor SOCS3 protein. As expected, ERK1/2 protein levels were selectively decreased in response to treatment with the p44/42MAPK inhibitor, indicating specific inhibition of the p44/42MAPK pathway. Bcl2 protein levels in BaF3/Bcl2 cells remained unchanged in the presence or absence of the p44/42MAPK inhibitor. As controls, inhibitors of p38MAPK (SB203580), AKT (Triciribine), and PI3K (LY294002) were evaluated and none were found to affect SOCS3 protein levels in BaF3/Bcl2 cells. These data indicate that Bcl2-associated induction of SOCS3 in B cells involves activation of the p44/42MAPK cell signaling pathway and is independent of STAT3 activation. These studies illustrate a novel and previously unappreciated Bcl2-associated signaling pathway involving SOCS3 induction in B cells that may play an important role in B cell biology and in Bcl2-associated hematologic malignancies.

Attenuation of retinal vascular development and neovascularization during oxygen-induced ischemic retinopathy in Bcl-2−/− mice

Bcl-2 is a death repressor that protects cells from apoptosis mediated by a variety of stimuli. Bcl-2 expression is regulated by both pro- and anti-angiogenic factors; thus, it may play a central role during angiogenesis. However, the role of bcl-2 in vascular development and growth of new vessels requires further delineation. In this study, we investigated the physiological role of bcl-2 in development of retinal vasculature and retinal neovascularization during oxygen-induced ischemic retinopathy (OIR). Mice deficient in bcl-2 exhibited a significant decrease in retinal vascular density compared to wild-type mice. This was attributed to a decreased number of endothelial cells and pericytes in retinas from bcl-2−/− mice. We observed, in bcl-2−/− mice, delayed development of retinal vasculature and remodeling, and a significant decrease in the number of major arteries, which branch off from near the optic nerve. Interestingly, hyaloid vessel regression, an apoptosis-dependent process, was not affected in the absence of bcl-2. The retinal vasculature of bcl-2−/− mice exhibited a similar sensitivity to hyperoxia-mediated vessel obliteration compared to wild-type mice during OIR. However, the degree of ischemia-induced retinal neovascularization was significantly reduced in bcl-2−/− mice. These results suggest that expression of bcl-2 is required for appropriate development of retinal vasculature as well as its neovascularization during OIR.