Double Expression Research Articles

Results of a Phase Ib Study of Venetoclax Plus R- or G-CHOP in Patients with B-Cell Non-Hodgkin Lymphoma

Results of a Phase Ib Study of Venetoclax Plus R- or G-CHOP in Patients with B-Cell Non-Hodgkin Lymphoma

Impact of Serum Albumin in the Prognosis of Myc-Positive Aggressive B-Cell Lymphomas: Potential for a Novel Prognostic Score

Background: MYC positive double hit lymphomas (DHL) (presence of c-myc and BCL2 or BCL6 rearrangements by florescence in situ hybridization (FISH) analysis) and double protein expressor lymphomas (DEL) (protein expression by immunohistochemistry (IHC)) represent a subset of diffuse large B cell lymphomas (DLBCL) with poor prognosis. Several prognostic scores systems have been applied such as the International Prognostic Index (IPI) and Revised IPI (R-IPI) but the most effective score remains to be elucidated. We explored the role of serum albumin (SA) in the prognosis of DHL/DEL as previously shown in de novo DLBCL (Dalia 2013) and developed a simple prognostic model that could predict clinical outcomes.Methods: We included 91 patients from Moffitt Cancer Center from January 2008 to December 2015 diagnosed with DHL/DEL and performed a retrospective chart review after obtaining institutional review board (IRB) approval. Clinical data included SA, hemoglobin (Hb), age, IPI, treatment type, stage, bulky disease (>10 cm), extranodal involvement, cell of origin (COO) by Hans criteria, relapse and vital status. SPSS and Prism GraphPad were used to perform multivariate analysis (MV) and Kaplan-Meier (K-M) to evaluate statistical significance and survival outcome.Results: Ninety-one patients (DHL= 53, DEL= 38) were included in the analysis. The median age was 63 years (18-87) with 36 (39.56%) females. There was stage III/IV disease in 80.22%, bulky disease in 48.35%, extranodal involvement in 39% of the patients and 15.38% patients had CNS involvement. With a median follow-up of 15.2 months, the median overall survival (OS) was 18.2 months (95%CI 11.9-24.5). MV analysis showed that SA ≤ 3.7g/dL (HR 3.85, 95% CI 1.67-8.81, p = 0.001), Eastern Cooperative Oncology Group (ECOG) performance status (PS) ≥2 (HR 2.28, 95% CI 1.00-5.16, p = 0.049) and Ann Arbor Stage III/IV (HR 2.58, 95% CI 1.19-5.62, p = 0.016) were statistically significant associated with poor OS. We devised the SEA score (Stage, ECOG and Albumin) as follows in 3 risk categories: low risk (LR):0-1 points, intermediate risk (IR): 2 points, high risk (HR): 3 points. K-M OS curves are shown in Figure 1. The median OS for LR, IR and HR were 30.6 (95% CI 13.2 - 48.1), 13.2 (95% CI 7.3-19.1), 7.4 (95% CI 4.2-10.6) months, respectively, p < 0.001.We further explored the impact of SEA score separately in DHL and DEL subgroups. The median OS of DHL was significantly lower than DEL (29.40 vs. 13.30 months, respectively, p = 0.027, Figure 2A). SEA score was able to stratify both DEL and DHL subgroups accurately (Figure 2B and Figure 2C). DEL cohort had significantly greater OS compared to DHL in all risk groups (LR: 44.0 vs. 17.6 months, IR: 13.2 vs. 9.5 months, HR: 8.8 vs. 6.05 months; p< 0.001).Conclusions: DHL/DEL is a heterogeneous disease with a poor OS in this study. We demonstrated that SA<3.7 g/dl is independently associated with poor OS. Hypoalbuminemia could represent a surrogate of comorbid status and worse biology in DHL/DEL patients. SA based prognostic system accurately identified three distinct risk categories. SA could be considered in the risk stratification of DHL/DEL. [Display omitted] DisclosuresShah:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Baxalta: Membership on an entity's Board of Directors or advisory committees; Incyte: Research Funding; Rosetta Genomics: Research Funding; Bayer: Honoraria; Pfizer: Honoraria. Bello:Seattle Genetics: Speakers Bureau; Gilead: Speakers Bureau. Locke:Kite: Membership on an entity's Board of Directors or advisory committees. Chavez:Janssen: Speakers Bureau.

Cell of Origin and Double Protein Expression As Predictors of Outcome after Autologous Stem Cell Transplant for Patients with Relapsed/Refractory Diffuse Large B-Cell Lymphoma (DLBCL) Who Undergo Intensive Consolidation Therapy (ICT) and Autologous Stem Cell Transplant (AutoSCT)

Introduction: For patients with relapsed or refractory DLBCL with chemosensitive disease after 2nd line (salvage treatment), AutoHCT is considered the standard of care. Risk factors for progression following AutoHCT include primary refractory disease and early relapse (<12 months after initial chemotherapy. In our institution, we have previously shown that a course of ICT given prior to AutoHCT in patients with these high-risk features results in improved outcomes in DLBCL and other lymphomas (Damon et al, BMT 2008; 42(10):649-57). More recently, cell of origin (COO) subtype and double myc and bcl2 protein expression (DPP) status have been shown to predict outcome to front line treatment but data in the relapsed setting are lacking. We hypothesized that our approach of ICT followed by AutoHCT will increase the likelihood of complete remission (especially in patients with high-risk clinical or biologic features) and improve progression free survival (PFS) following AutoHCT.Methods: Consecutive patients who underwent ICT followed by autoHCT for relapsed/refractory DLBCL at our institution between 2005-2015 and had available tissue samples for review were included in our analysis. ICT was applied per institutional standard to patients with apriori identified high-risk features as described below. This chemotherapy step is utilized for stem cell mobilization following evidence of chemosensitivity and prior to undergoing AutoHCT. COO was determined using the Hans algorithm and DPP status using the Johnson criteria. Following CHR approval, patient's charts and EMR were reviewed to ascertain baseline variables and outcome data. Response was determined using radiologic (CT) and metabolic (PET) imaging for all patients at the time of transplantation. Survival probability was estimated by the Kaplan-Meier method. Comparisons were made using the X2 test and Fisher's exact test, where appropriate.Results: A total of 35 patients were included in this analysis. Out of those, 30 patients received a high-dose etoposide and ara-C-based ICT regimen as described by Damon et al. 13/30 (43.3%) were female, 17/30 (56.7%) were male. Median age at transplant was 56 (range 34-68). The high risk features that lead to this treatment were: early (<12 months) relapse (9/30, 30%) or refractory disease (13/30, 43.3%)to front line therapy or other (8/30, 27.7%). In all, 16/30 (53%) patients achieved CR at the end of salvage therapy, with this proportion increasing to 26/30 (86.7%) CR after ICT. Eventually, 24/30 (80%) of patients were able to continue on to AutoHCT, 23/30 (76.7%) with CBV, 4/30 (13.3%) with BEAM, and 3/10 (10.0%) other. With a median follow up of 20 months, 66% of patients are progression-free at 24 months (Figure 1). In our cohort, 8/30 (27%) were germinal center B-cell origin subtype (GCB), 19/30 (63.3%) were non-GCB subtype and 3 (10.0%) could not be identified. In all, 8/30 (26.7%) were double protein positive, 18 (60.0% were double protein negative and 4 (13.3%) could not be determined. Notably, 15.8% of patients with non-GCB were double protein expressors, compared to 37.5% of patients with GCB (Fisher’s exact p=0.06). Neither COO nor DPP were associated with response rates prior to AutoHCT (p=0.6 and p=0.17 respectively) or with PFS (p=0.32 and p=0.72 respectively and Figures 2a and 2b).Conclusions: Even though COO and DPP status are associated with inferior outcomes in the front-line setting of patients with DLBCL, data in the relapsed setting are limited. In a cohort of high-risk patients with relapsed DLBCL, we confirmed our previously described excellent outcomes with ICT in terms of increased CR rates prior to AutoHCT and a high rate of 2-year PFS. Moreover, while numbers are small, our study suggests that ICT leads to similar outcomes regardless of cell of origin or double protein expression status. [Display omitted] [Display omitted] [Display omitted] DisclosuresMartin:Amgen: Research Funding; Sanofi: Research Funding.

High Risk Diffuse Large B Cell Lymphoma: A Comparison of Aggressive Subtypes Treated with Dose Adjusted Chemotherapy-the University of Texas MD Anderson Experience

Background: Diffuse large B cell lymphoma (DLBCL) is the most common type of non Hodgkin lymphoma (NHL).Nearly 50% of high-risk DLBCL patients are not cured with standard rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (RCHOP). High risk DLBCL may be defined as double hit lymphoma (DHL, translocation of MYC and BCL2 or BCL6), double expressor lymphoma (DEL, over expression of MYC and BCL2), high risk international prognostic index (IPI) of 3-5, high Ki-67, and non-germinal center subtype (non-GCB). The majority of DHL cases occur in the GCB subtype, as opposed to the majority of DEL cases which occur in non-GCB. Hence we sought to compare different high risk subsets treated with dose-adjusted etoposide, doxorubicin, cyclophosphamide, vincristine, prednisone and rituximab (DA) EPOCH-R. In single arm phase II clinical trials, dose adjusted (DA) EPOCH-R has shown promising results, with potential greater efficacy in the GCB subtype in subset analyses (Wilson et al, Hematologica 2012). A randomized phase III study comparing RCHOP with (DA) EPOCH-R in newly diagnosed DLBCL has completed accrual, with highly anticipated results due in late 2016.Methods: We conducted a retrospective reviewof all consecutive, newly diagnosed DLBCL patients treated with (DA) EPOCH-R at MD Anderson Cancer Center from 2010 to 2014. Eligible patients were 18 years or greater, had high-risk DLBCL as determined by the treating physician, and had available data of treatment and response. The cell of origin subtype was determined by immunohistochemistry using Hans algorithm, and MYC and BCL2 positivity were defined as BCL2 positive in at least 70% and MYC positive in at least 40% of cells. DHL was defined as rearrangement of MYC and BCL2 or BCL6 by fluorescent in situ hybridization. The objectives were to analyze demographic, prognostic, and treatment variables in comparison with clinical response and survival outcomes in three sub groups which included 1. DHL (GCB) 2. DLBCL without MYC and BCL2 expression (GCB), and 3. DEL (GCB and non GCB). Complete response (CR), overall survival (OS) and progression free survival (PFS) were calculated using standard methods. Statistical analysis was done using Fishers exact test or Chi-square test / Kruskal-Wallis test. Kaplan-Meier method was used for time-to-event analysis including overall survival and progression free survival. The Log-rank test was used to evaluate the difference in time-to-event endpoints between patient groups.Results: We identified 233 high risk DLBCL patients treated with (DA) EPOCH-R. After filtering the data to identify patients which were included in our three groups, we identified 22 patients with DHL (GCB), 46 patients with non DEL (GCB), and 16 with DEL. The demographic features and outcomes are mentioned in the table 1 below. The DHL group had more frequent bone marrow (BM) involvement, and the DHL and DEL groups were more frequently age >60 years and high IPI in comparison to the non DEL GCB group. The CR rate, OS and PFS at 1 year were not significantly different between these three groups. Figure 1 highlights the OS (A) and PFS (B) results of each group.Conclusions: (DA) EPOCH-R is highly effective in patients with subsets of patients with high-risk DLBCL and may be able to overcome prognostic factors which have been shown to be adverse with RCHOP therapy. The results of this retrospective study suggest that OS in DHL, DEL and non DEL (GCB) are not statistically different. Hence, intensive chemotherapy with (DA) EPOCH-R could be considered as a frontline treatment option for patients with high risk DLBCL, pending further confirmation in randomized trials. [Display omitted] [Display omitted] DisclosuresOki:Novartis: Research Funding. Fowler:Infinity: Consultancy, Research Funding; Roche: Consultancy, Research Funding; TG Therapeutics: Consultancy; Celgene: Consultancy, Research Funding; Jannsen: Consultancy, Research Funding; Gilead: Research Funding. Wang:Pharmacyclics: Research Funding; Juno Therapeutics: Research Funding; Acerta Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; BeiGene: Research Funding; Kite Pharma: Research Funding; Onyx: Research Funding; Asana BioSciences: Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Fayad:Seattle Genetics: Consultancy, Research Funding. Westin:ProNAi: Membership on an entity's Board of Directors or advisory committees; Spectrum: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Chugai: Membership on an entity's Board of Directors or advisory committees.

Myc and Bcl2 Overexpression and Traslocation Assessed By Immunohystochemistry (IHC) and FISH: Retrospective Analysis in a Series of De Novo DLBCL Homogeneously Treated with Rituximab-CHOP

Introduction: MYC and BCL2 overexpression assessed by IHC and rearrangement detected by FISH are important prognostic factor in Diffuse Large B-Cell Lymphoma (DLBCL). Double Hit Lymphoma (DHL) patients have a poor prognosis with conventional therapy and Double expressor Lymphoma (DE) have worse outcome compared with conventional DLBCL although data are controversial. Aimed at a better knowledge of this issue, we performed a retrospective analysis to determine prevalence and outcome of Single Hit Lymphoma (SHL), DHL and DE in patients with de-novo DLBCL treated with Rituximab and CHOP.Methods: de novo DLBCL treated with R-CHOP between January 2003 and December 2013 were included in the study. BCL2 and BCL6 expression were evaluated by IHC at diagnosis while MYC expression was retrospectively investigated with Tissue Micro Arrays (TMA) technique; cases were considered positive for MYC, BCL2 or BCL6 expression by IHC if >40%, >40% or >25% of cells stained positive, respectively. FISH analysis for MYC and BCL2 rearrangements were performed with dual color break apart probes on TMA. PFS and OS were estimated with Kaplan-Meier method and compared between groups with the Cox model.We further evaluated in this series the IHC score proposed by Botto et al. (Blood 2014 124:2964) in DLBCL. The score was based on the assessment in IHC of the expression of MYC, BCL2 and BCL6. The three variable contributed with different risk in the multivariate analysis and an IHC sum additive score of 0-5 was calculated proportionally to the coefficient estimated (coefficient [Log hazard ratio] 0.92 for MYC+, 0.73 for BCL2+ and 0.48 for BCL6-), assigning an individual risk of 2 points for MYC or BCL2 positivity and 1 point for BCL6 negativity. Patients were stratified in three different risk groups; Low risk (0-1 point), Intermediate risk (2 points) and High risk (≥3 points).Results: Of a total of299 DLBCL screened, 267 were evaluable for survival analysis; median age was 65 years (range 20-90). 154 patients had complete immunohystochemical data and 101 were fully investigated by IHC and FISH. No significant differences in clinical presentation or in the outcome were seen between patients with or without available histologic tissue for IHC and FISH. Among 154 patients with complete IHC data we found 12 (8%) DLBCL without expression (DLBCL), 96 (62%) Single expressor (SE), 46 DE (30%). With a median follow up of 60 months, 5-year PFS rates were: DLBCL 90%, SE 60% and DE 43% respectively (fig 1) (HR 8.25 (95% CI: 1.12 -60.99) p 0.039); 5ys OS rates were 91%, 68% and 57% respectively (HR 5.72 (95% CI: 0.77 - 42.82) p 0.08).Applying the prognostic model (adjusted for IPI and age) defined in our previous pilot study we recorded 12/154 patients with low risk score, 61/154 with intermediate and 81/154 patients in high risk group. 5y-PFS rates were 91% vs 67% vs 45% (p=0.014) respectively (HR 1.74 (95% CI: 1.12 -2.69) p 0.014). Among 101 patients investigated by FISH we recorded 10 SHL (10%) and 8 DHL (8%). Clinical characteristics were superimposable among DLBCL, SHL and DHL with a prevalence of non GCB phenotype in DE group (56%). Among 101 patients fully investigated for FISH and IHC, 38 patients had MYC overexpression by IHC; 11 of them had also a MYC translocation. We found 3 cases with MYC rearrangement without protein overexpression. With a median follow up of 60 months PFS in DLBCL, SHL and DHL was 65%, 58% and 25% respectively (fig. 2); 5 ys OS was 70%, 77% and 25% respectively. The worse prognosis of DHL was statistically significant with an HR of 3.3 (95% CI: 1.37- 7.94, p 0.008) in terms of PFS and 3.9 (95% CI:1.59- 9.52 p 0.003) in terms of OS.Conclusion: Our data confirmed that our IHC prognostic score based on MYC, BCL2 and BCL6 expression, is a simple, reproducible and valid prognostic assessment that identify three groups with a different outcome in a large cohort of DLBCL. Moreover these data confirm intermediate prognosis for patients with DE lymphoma and poor prognosis of DHL treated with conventional chemoimmunotherapy. [Display omitted] [Display omitted] DisclosuresChiappella:Roche: Speakers Bureau; Celgene: Speakers Bureau; Janssen-Cilag: Speakers Bureau; Teva: Speakers Bureau; Pfizer: Speakers Bureau; Amgen: Speakers Bureau. Cavallo:Celgene: Honoraria; Onyx: Honoraria; Janssen-Cilag: Honoraria. Vitolo:Celgene: Honoraria; Gilead: Honoraria; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria; Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Phase 1 Trial Testing Single Agent CUDC-907, a Novel, Oral Dual Inhibitor of Histone Deacetylase (HDAC) and PI3K: Initial Assessment of Patients with Relapsed or Refractory (RR) Diffuse Large B-Cell Lymphoma (DLBCL), Including Double Expressor (DE) Lymphoma

DLBCL, the most common lymphoid malignancy in adults, is an aggressive form of non-Hodgkin lymphoma (NHL) with significant heterogeneity in terms of gene expression, prognosis and response to treatment. Certain histone modifications and HDAC expression patterns have been implicated in the pathobiology of DLBCL and other cancers. Aberrant PTEN/PI3K/AKT signaling is also frequently observed in a variety of human cancers including DLBCL, where loss of PTEN activity and/or activating mutations in PI3K and AKT have been shown to reduce apoptosis and promote cell proliferation. Synergistic anti-tumor effects achieved with HDAC and PI3K inhibitor combinations in various DLBCL xenograft models have provided a strong rationale for testing a highly potent dual HDAC and PI3K inhibitor, CUDC-907. Significant anti-tumor effects have been observed across a wide range of DLBCL models exposed to CUDC-907, including the U2932 ABC DLBCL model that harbors BCL2 translocation and over-expresses cMYC and BCL6, and the WSU-DLCL-2 GCB DLBCL model that harbors EZH2 mutation. These disease mechanisms and preclinical data have led to testing of CUDC-907 in an ongoing Phase 1 trial in patients (pts) with various hematologic cancers.Sixty-three heavily pre-treated pts with lymphoma or multiple myeloma have received CUDC-907 in 21-day cycles according to once daily (QD), intermittent (BIW or TIW), or five days on/two days off (5/2) dosing schedules. CUDC-907 was escalated in 30 mg increments such that pts received 30-60 mg QD, 60-150 mg intermittently, or 60 mg 5/2. A dose expansion using the 60 mg 5/2 dose and schedule is ongoing. Thus far, CUDC-907 has demonstrated a manageable side effect profile and sustained clinical efficacy, particularly in the subset of pts with RR DLBCL.The most common treatment-related adverse events (AEs) were diarrhea (54%; 5% Grade ≥3), fatigue (37%; 3% Grade ≥3), nausea (22%) and thrombocytopenia (18%; 14% Grade ≥3), with dose limiting toxicities of hyperglycemia (QD and BIW schedules) and diarrhea (QD and TIW schedules). Side effects were reversible and manageable. Among 11/18 subjects with RR DLBCL who were evaluable for disease response, 6/11 (55%) achieved objective responses (2 CRs and 4 PRs); lasting a median of 119 days (range: 48 - 354+). Of 7 pts with sufficient tissue, 3 response-evaluable pts were found to over-express MYC and BCL2 by IHC, meeting criteria applied to "double-expressor" (DE) DLBCL. On CUDC-907 monotherapy, 2/3 confirmed pts with DE DLBCL have attained objective responses: 1 ongoing CR (followed by autologous stem cell transplant) and 1PR (lasting 132 days). The third response-evaluable pt with DE DLBCL has experienced lengthy stabilization of disease (171+ days).Signals emerging from preclinical and clinical studies suggest that pts with DLBCL, including those with particularly aggressive disease, may derive benefit from treatment with CUDC-907. Enrollment of pts with RR DLBCL is ongoing into the expansion phase of the Phase 1 trial that is testing CUDC-907 on the 60 mg 5/2 RP2D as monotherapy and in combination with rituximab. Tissue obtained from pts with RR DLBCL treated with CUDC-907 will be assessed for MYC, BCL2, and other genetic aberrations that will be correlated with clinical outcomes observed in the trial. A Phase 2 trial of CUDC-907 in RR NHL is currently being planned, with emphasis on MYC aberrations. DisclosuresYounes:Celgene: Honoraria; Incyte: Honoraria; Janssen: Honoraria; Seattle Genetics: Honoraria, Research Funding; Bayer: Honoraria; Bristol Meyer Squibb: Honoraria; Novartis: Research Funding; Sanofi-Aventis: Honoraria; Johnson and Johnson: Research Funding; Curis: Research Funding; Takeda Millenium: Honoraria. Berdeja:Acetylon: Research Funding; Onyx: Research Funding; Abbvie: Research Funding; Curis: Research Funding; Celgene: Research Funding; Takeda: Research Funding; MEI: Research Funding; Novartis: Research Funding; Janssen: Research Funding; BMS: Research Funding; Array: Research Funding. Flinn:Celgene Corporation: Research Funding. Clancy:Curis: Employment, Equity Ownership. Ma:Curis: Employment, Equity Ownership. Sun:Curis: Employment, Equity Ownership. Tian:Curis: Employment, Equity Ownership. Wang:Curis: Employment, Equity Ownership. Viner:Curis: Employment, Equity Ownership.

High Dose Chemotherapy with Autograft in First Relapse May Overcome the Poor Prognosis of Diffuse Large B-Cell Lymphoma Patients with MYC/BCL2 Co-Expression

The immunohistochemical (ICH) co-expression of MYC and BCL2 (double expressors, DE) has been emerging as a strong and reproducible risk factor in diffuse large B cell lymphoma (DLBCL) patients (pts) treated with R-CHOP. Our aim was to analyze the prognostic value of the co-expression in DLBCL pts treated with high-dose chemotherapy followed by autologous stem cell transplantation at first relapse.In this retrospective study, we analyzed a cohort of 64 young [median age 53, (range18-70)] and/or fit DLBCL pts treated between 2003 and 2015 at the Istituto Nazionale dei Tumori Milano. All pts relapsed at median time of 4.9 months after a Rituximab-based chemotherapy (48 R-CHOP-like and 16 other intensive regimens). All pts were considered fit and eligible for high-dose chemotherapy salvage treatment. Tumor samples were analyzed by ICH for MYC and BCL2 (40%/70% threshold). Germinal B-Cell (GBC) and Activated B-Cell (ABC) DLBCL classification was done with ICH as established by the Hans algorithm. Pts characteristics at relapse were: refractory: 37/64 (58%), Ann Arbor stage≥3: 42/64 (65%), IPI score≥2: 26/60 (43%); Extranodal disease: 37/64 (58%); Bulky (defined as max diameter ≥5 cm): 29/64 (45%); CNS involvement: 8/64 (12.5%).Twenty-nine of 64 (45%) pts responded to salvage high-dose therapy (CR,PR) and 25 of them underwent autologous stem cell transplantation (ASCT). Among 39 (61%) refractory pts to first salvage intensive chemotherapy approaches, 11 (28%) responded after third or fourth chemo-immunotherapy salvage lines and underwent ASCT as well. Overall the reasons for not receiving ASCT were: refractory disease (n=20), poor mobilization (n=3), the occurrence of significant infection or toxic complications during the salvage therapy (n=4) or patient decision (n=1). ICH MYC and BCL2 positive expression was observed in 31 (48%) and 39 (61%) pts respectively. Among them, 17 (26%) were characterized by BCL2 and MYC co-expression. According with Hans algorithm, 23 (56%) and 18 (44%) DLBCL pts were classified as GBC and ABC-DLBCL. DE pts did not show any significant differences compared to others in terms of age, IPI score, presence of extranodal disease, BM infiltration, CNS involvement at relapse, refractory disease or relapse occurrence less than 1 year after first line therapy, and proportion of ABC-DLBCL subtype. Considering the whole pts cohort 5-years progression free survival (PFS) and overall survival (OS) were 27.3% (95% CI, 21.1%-33,4%) and 40.6% (95% CI, 33.2%-48%) respectively. In univariate analysis, pts with disease relapsed less than 1 year after first line therapy were characterized by a significant worse outcome in terms of 5-years PFS [11.5% (95% IC 6.1%-16.9%) vs 72.2% (95% IC 60%-84.4%] and OS [24.5% (95% IC 16.9%-32.1%) vs 84.4% (95% IC 74%-94.8%)] (p=0.0001 and p=0.0009 respectively). Similar results were observed comparing pts with refractory disease to first line therapy with others [5-years PFS 11.7% (95% IC, 5.9%-17.5%) vs 48.2% (95% IC, 37.1%-59.3%)] and 5-years OS [19.9% (95% IC, 11.8%-28%) vs 67.9% (95% IC, 57.6%-78.2%)] (p=0.0003 and p=0.001 respectively). Other variables at first relapse associated with worse outcome in terms of PFS and OS were IPI score ≥2 and presence of bulky disease. In contrast, DE patients did not shown any significant differences compared to other patents in term of 5-years PFS [39.7 (95% IC, 27.5-51.9) vs 25.1 (95% IC, 18.4-31.8), p=0.5] and 5-years OS [40.8 (95% IC, 32.3-49.3) vs 22.2 (95% IC, 5-39.4), p=0.8]. Furthermore singularly ICH MYC or BCL2 expression did not influence neither PFS nor OS.These data suggest that salvage programs with high dose chemotherapy with autograft in first relapse may overcome the previously reported poor prognosis associated with ICH MYC/BCL2 co-expression. The most significant and robust prognostic factor among DLBCL pts in first relapse is still represented by refractoriness and the time of relapse. DisclosuresNo relevant conflicts of interest to declare.

The Prognostic Role of Cell of Origin Profile and Myc Expression Assessed By Immunohistochemistry in Young High-Risk Patients with Diffuse Large B-Cell Lymphoma (DLBCL): Results of First-Line Randomized BIO-DLCL04 Trial of Fondazione Italiana Linfomi (FIL)

Background. The prognostic role of cell of origin profile (COO) assessed by immunohistochemistry (IHC) is controversial in Rituximab era. FIL conducted a phase III randomized trial aimed at investigating the benefit of intensification with high dose therapy plus autotransplant compared to R-dose-dense therapy as first line in young DLBCL at poor risk (aa-IPI 2-3). Clinical results were reported (Vitolo, ASH 2012). The aim of BIO-DLCL04 was to correlate the biological markers with PFS. Patients and Methods. From 2005 to 2010, 412 untreated DLBCL at aa-IPI 2-3 were enrolled. Central histology revision was mandatory and 13 patients were excluded due to different histologies. Biological markers were analyzed on DLBCL NAS; COO analysis was performed by IHC and cases were classified in germinal center (GC) and non-GC according to Hans' algorithm; COO determined by gene expression profile using the NanoString® nCounter® Analysis System based on 20-gene assay (Lymph2Cx) using formalin fixed paraffin embedded tissue is ongoing; BCL2, BCL6 and MYC anomalies were tested by IHC; final analysis by fluorescent in situ hybridization (FISH) is ongoing. Cases were deemed positive if at least 30% of lymphoma cells were stained with each antibody (with the exception of at least 40% for MYC). Results. At the time of this analysis, 223 DLBCL NAS were analyzed: 131 non-GC and 92 GC; BCL2, BCL6 and MYC anomalies were tested in 196, 74 and 107 cases respectively. Clinical characteristics for non-GC vs GC were: median age 51 years for both, male 49% vs 45%, aa-IPI 3 15% vs 25%, bone marrow involvement (BM) 16% vs 24%. R-HDC was performed in 45% of non-GC patients and in 49% of GC. Complete response was recorded in 105 (80%) non-GC patients and in 62 (67%) GC. At a median follow-up of 49 months, the 3-year PFS for non-GC vs GC was 75% (95% CI: 67-82) vs 57% (95% CI: 46-67) with crude hazard ratio, HR 0.55 (0.35-0.87), p.01 and adjusted (for age, gender, aa-IPI, BM) aHR 0.56 (0.35-0.88), p.013. No significant differences by treatment were reported. Overexpression of MYC by IHC had a relevant prognostic impact, with aHR 1.84 (0.99-3.44), p.054. By IHC, 3-years PFS for double negative vs single BCL2 or MYC overexpression vs double positive, was 85% vs 68% vs 51% respectively, with an aHR for double expressors compared to double negative of 3.91 (1.13-13.53), p.031. At the time of the present report, FISH analysis was conducted in 88 cases: 43 were triple negative, 37 single hit and 8 double/triple hit. By FISH, 3-years PFS for triple negative vs single hit vs double/triple hit was 74% vs 84% vs 25% respectively, with an aHR for double/triple hit compared to triple negative of 5.73 (2.05 to 16.02), p.001. Conclusions. In conclusion, with the limit of the analysis performed by IHC based on Hans' algorithm, BIO-DLCL04 showed an unexpected better outcome for non-GC compared to GC, irrespective of treatment arm. The ongoing analysis conducted by Nanostring will be more informative. The overexpression of MYC was an unfavourable risk factor, mainly if associated with BCL2 overexpression, irrespective of type of treatment. Moreover, double/triple hit patients represent a subgroup with extremely poor prognosis. High dose therapy plus autotransplant was not able to reverse the inferior outcome of neither double expressors nor double hit patients and new strategies are deemed for these poor prognosis patients. DisclosuresNo relevant conflicts of interest to declare.

Identification of a Meta-Gene Network Associated with Metformin Sensitivity and Clinical Outcomes in Double Hit and Double Expressor Lymphomas

▪BackgroundThe "double hit" (DH) lymphomas that harbor a c-myc mutation and BCL2 translocation, or "double protein expressor" (DP) lymphomas that over-express c-myc and BCL2 proteins in the absence of a detectable mutation, have amongst the worst clinical outcomes as compared to patients with diffuse large B-cell lymphomas (DLBCL) that lack upregulation of the c-myc oncogene. Metformin can down-regulate translation of c-myc, making it an appropriate anti-cancer drug to explore in c-myc+ lymphomas. Furthermore, amethod to identify DH/DP patients most likely to benefit from metformin treatment has clinical relevance.MethodsWithin a publicly available gene expression array data set of R-CHOP treated DLBCL (n=232; GSE10846), a subset of DH/DP patients were defined as having above median expression of myc and BCL2 and below median expression of BCL6 as previously published by Horn et al. Survival analysis, significance analysis of microarrays (SAM) and gene set analysis (GSA) were performed characterizing the clinical, individual gene and biological ontology differences between DH/DP and non-DH/DP populations. Expression array data from a study testing metformin effects on THP-1 monocyte cells was reanalyzed using SAM and GSA as well. Changes in individual gene expression and overlapping ontological themes common to both GSA analyses of metformin effects on THP-1 cells and DH/DP characterization were identified. Genes with differential expression (DE) in both groups were evaluated topologically using a protein-protein interaction database to determine if any gene products had previously observed direct interactions. Network community detection identified tightly coupled signaling modules linking co-expression to mechanism. The resulting metformin-DH/DP network metagene was evaluated by k-means, clustering tumor samples into two groups over the metagene members in an independent data set of R-CHOP treated DLBCL patients (n=249; GSE32918) with differences in overall survival (OS) determined by log-rank.ResultsOf the 232 DLBCL patients treated with R-CHOP, 26 fit the criteria for DH/DP and had significantly lower OS (HR = 2.96; p < 0.001). In DH/DP tumors, 2780 genes had DE (2208 up-regulated; 572 down-regulated), enriched for biological processes related to transcription, metabolism and cytokine production and down-regulated for processes related to immune response, cell signaling, vascular development and proliferation (Fig. 1A). Analysis of metformin treated THP-1 cells relative to control identified 7123 genes with DE. Biological themes common to metformin treatment and DH/DP specific biology were identified including mitochondrial biogenesis, alternate splicing, and hormone secretion (Fig. 1A-B; highlighted in red). The intersection of genes with DE in metformin treated and DH/DP data sets identified 102 genes with direct interaction within a protein interaction network. Of the 19 communities detected by analyzing the resulting network topology, 3 showed significant correlation to survival in the GSE10846 data set (Fig. 2A, in red), forming a metformin-DH/DP metagene (Met-DH/DP-MG; n = 29 genes total). This metagene was validated by applying it to an independent cohort of R-CHOP treated DLBCL patients (n = 249), demonstrating 2 cluster groups (cluster 1, n=178; cluster 2, n=71; Fig. 2B) with differences in OS (HR = 1.61; p < 0.001; Fig. 2C).ConclusionWe have identified a metagene of interacting proteins associated with both metformin therapeutic effect and OS in DH/DP patients. This offers a potential method for selecting patients most likely to benefit from metformin therapy and identifies mechanistic avenues by which metformin treatment may specifically benefit DH/DP patients. As such, in vitro studies using DH cell lines and a phase I/II clinical trial exploring chemo-immunotherapy with metformin as an adjunct in DH/DP lymphomas are underway. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Clinical Significance of Co-Expression of MYC and BCL2 Protein in Advanced Diffuse Large B-Cell Lymphoma Treated with a Dose-Intensified Immunochemotherapy

BackgroundRecent studies have shown that the concurrent expression of MYC and BCL2 protein evaluated by immunohistochemistry (IHC) in patients with de novo diffuse large B-cell lymphoma (DLBCL) is associated with worse survival when treated with standard R-CHOP, but the effect of intensive chemotherapies for such patients is unknown. Thus, we evaluated the impact of the co-expression of MYC and BCL2 protein among patients with advanced DLBCL, who were treated with a dose-intensive immunochemotherapy followed by up-front autologous stem cell transplantation (ASCT).Patients and MethodsThis is a retrospective analysis of patients with de novo DLBCL, who were categorized into high/high-intermediate risk by the age-adjusted International Prognostic Index (aaIPI). They were consecutively treated with the R-Double-CHOP regimen, consisting of rituximab (375 mg/m2, day -2), cyclophosphamide (750 mg/m2, day 1, 2), doxorubicin (50 mg/m2, day 1, 2), vincristine (1.4 mg/m2 [maximum 2.0 mg/body], day 1) and prednisolone (50 mg/m2, day 1-5) followed by consolidative high-dose chemotherapies at our institution from 2001 to 2013. MYC and BCL2 protein were measured by IHC assay using formalin-fixed paraffin-embedded tissue specimens for all available cases. Cut-off values of positivity for MYC and BCL2 protein were set as 40% and 50% of stained tumor cell, respectively. Lymphomas showing concurrent positivity for MYC and BCL2 protein were defined as "Double expressor lymphoma (DEL)".ResultsA total of 40 patients with a median 53-years (range 19-68) of age were analyzed. Twenty-one patients were at high risk and the other 19 patients were at high-intermediate risk by aaIPI. Cell of origin (COO) subtypes classified by Hans algorithm consisted of 14 germinal center B-cell (GCB) type and 26 non-GCB type. Totally, 10 (25%) patients were categorized into DEL. The overall response (OR) and the complete response (CR) rates to R-Double-CHOP for all patients were 93% and 83%, respectively. The OR and the CR rates were not significantly different between the DEL group and the non-DEL group (100% vs 90%, and 80% vs 83%, respectively). The proportion of patients proceeding to ASCT was not significantly different among these groups (50% vs 60%). With a median 52 months (range 3-155) of follow-up, the 3-year progression-free survival (PFS) and the overall survival (OS) rates for all patients were 55% and 72%, respectively (Figure a, b). Both the PFS and the OS were significantly worse in the DEL group than in the non-DEL group (Figure c, d). As for aaIPI and COO subtyping, either high/high-intermediate risk or GCB/non-GCB subtype were not significantly associated with the outcome of PFS or OS.ConclusionThe concurrent expression of MYC/BCL2 protein in advanced DLBCL was associated with shorter remission duration and worse survival despite similar susceptibility to the treatment when a dose-intensive immunochemotherapy was applied. Our findings suggest that patients with advanced DEL may not benefit from dose-intensified therapies, and therefore need highly discrete strategies. [Display omitted] DisclosuresMiura:Astellas Pharma Inc.: Honoraria; Celgene K.K.: Honoraria; Sumitomo Dainippon Pharma Co., Ltd.: Honoraria; CHUGAI PHARMACEUTICAL CO. LTD: Honoraria; Kyowa Hakko Kirin CO., Ltd, Japan: Honoraria; Meiji Seika Pharma: Honoraria; Janssen Pharmaceutical K.K.: Honoraria. Hatta:Kyowa Hakko Kirin CO., Ltd, Japan: Honoraria; CHUGAI PHARMACEUTICAL CO. LTD: Honoraria; Celgene K.K.: Honoraria. Iriyama:Brystol-Myers K.K.: Honoraria. Takei:Kyowa Hakko Kirin CO., Ltd, Japan: Research Funding; Bristol-Myers K.K.: Research Funding; Nippon Kayaku Co.: Research Funding; Shionogi & Co.: Research Funding; Meiji Seika Pharma: Research Funding; Astellas Pharma Inc.: Research Funding; Janssen Pharmaceutical K.K.: Research Funding; TEIJIN PHARMA LIMITED: Research Funding; CSL Behring K.K: Research Funding; Japan Blood Products Organization: Research Funding; Sumitomo Dainippon Pharma Co.: Research Funding; TORII, PHAMACEUTICAL CO: Research Funding; Alexion Pharmaceuticals: Research Funding; YAKULT HONSHA CO., Ltd.: Research Funding; Taisho Toyama Pharmaceutical Co., Ltd.: Research Funding; TAIHO PHARMACEUTICAL CO., Ltd.: Research Funding; CHUGAI PHARMACEUTICAL CO. LTD: Research Funding.

Steroid receptor RNA activator protein (SRAP) expression as a prognostic factor in ER+ human breast tumors

The steroid receptor RNA activator protein (SRAP) is a newly described protein modulating the activity of multiple transcription factors including the estrogen receptor (ER). We have recently reported the immunodetection by Western blot of multiple SRAP peptides in breast tissue. High expression of these peptides, assessed by tissue micro-array (TMA) analysis, was associated with poor prognosis in patients whose primary tumors were ER positive (ER+). In such studies, it is recognized that intensity as well as specificity of the signal detected directly depends upon the antibody used as well as the position of the epitope recognized. To confirm the potential relevance of SRAP as a new prognostic factor, it is critical to establish whether similar results are obtained with independent antibodies. Two commercial anti-SRAP antibodies (742A and 743A), respectively, recognizing the N- and C-terminal extremity of the protein, were first used to analyze by Western blot SRAP expression in protein extracts from frozen breast tumor tissue sections. These antibodies were further used to investigate by immunohistochemistry (IHC) SRAP location in paraffin-embedded breast tumors. Comparative TMA analysis of 170 ER+ tumors was eventually performed in order to establish the potential associations existing between SRAP expression and clinical outcome. Multiple SRAP peptides were differentially detected by Western blot. Both antibodies led to similar nuclear and cytoplasmic staining in breast tissue section. A solid correlation was found (Spearman r = 0.46, P < 0.001) between 742A and 743A IHC scores. Results from both antibodies independently showed that dividing expression levels into lower 25 percentile, 26-75 percentile, and highest 25 percentile demonstrated a hazard ratio (HR) of 1.82 (P = 0.0042) for 742A antibody and 1.35 (P = 0.14) for 743A antibody. When both scores are combined, double high expressor (by 742A and 743A) was associated with a poor prognosis of breast-cancer-specific survival (Mantel-Cox: P = 0.005, HR = 2.24). Overall, our data suggest the existence in breast tumor tissue of multiple SRAP-like peptides. Assessing their expression in primary breast tumors can predict clinical outcome in ER+ breast cancer patients.

Multifunctional CD4+ T cells correlate with active Mycobacterium tuberculosis infection

Th1 CD4(+) T cells and their derived cytokines are crucial for protection against Mycobacterium tuberculosis. Using multiparametric flow cytometry, we have evaluated the distribution of seven distinct functional states (IFN-γ/IL-2/TNF-α triple expressors, IFN-γ/IL-2, IFN-γ/TNF-α or TNF-α/IL-2 double expressors or IFN-γ, IL-2 or TNF-α single expressors) of CD4(+) T cells in individuals with latent M. tuberculosis infection (LTBI) and active tuberculosis (TB). We found that triple expressors, while detectable in 85-90%TB patients, were only present in 10-15% of LTBI subjects. On the contrary, LTBI subjects had significantly higher (12- to 15-fold) proportions of IL-2/IFN-γ double and IFN-γ single expressors as compared with the other CD4(+) T-cell subsets. Proportions of the other double or single CD4(+) T-cell expressors did not differ between TB and LTBI subjects. These distinct IFN-γ, IL-2 and TNF-α profiles of M. tuberculosis-specific CD4(+) T cells seem to be associated with live bacterial loads, as indicated by the decrease in frequency of multifunctional T cells in TB-infected patients after completion of anti-mycobacterial therapy. Our results suggest that phenotypic and functional signatures of CD4(+) T cells may serve as immunological correlates of protection and curative host responses, and be a useful tool to monitor the efficacy of anti-mycobacterial therapy.

Expression and purification of non-glycosylated Trypanosoma brucei transferrin receptor in insect cells

The transferrin receptor of the parasite Trypanosoma brucei is a heterodimeric protein complex encoded by the 2 expression site-associated genes ( ESAGs) 6 and 7. ESAG6 is a heterogeneously glycosylated protein of 50–60 kDa modified by a glycosylphosphatidylinositol anchor at the C-terminus, while ESAG7 is a 40–42 kDa glycoprotein carrying an unmodified C-terminus. In order to determine whether glycosylation is necessary for dimer formation and ligand binding, the receptor was expressed in insect cells in the presence of tunicamycin. When insect cells were infected with recombinant ESAG6/ ESAG7 double expressor baculovirus and grown in the presence of tunicamycin, non-glycosylated forms of ESAG6 and ESAG7 of 46 and 36 kDa, respectively, were synthesized. The non-glycosylated ESAG6 and ESAG7 were capable of forming a heterodimer and of binding transferrin. This results shows that glycosylation is not necessary for synthesis of a functional T. brucei transferrin receptor.

Control of variant surface glycoprotein gene-expression sites in Trypanosoma brucei.

Trypanosoma brucei has 20 similar telomeric-expression sites for variant surface glycoprotein genes. Expression sites appear to be controlled at the level of transcription initiation, resulting in only one site being active at any time. Switching between expression sites occurs at a low rate. To analyse the switching mechanism, we used trypanosomes with two expression sites tagged with two different drug-resistance genes and selected these on agarose plates containing both drugs. Double-resistant clones arose at a low frequency of 10(-7) per cell, but these behaved as if they rapidly switched between the two tagged expression sites and lost double resistance in the absence of selection. Using in situ hybridization we found that only 10% of the double-resistant cells had two fluorescent spots corresponding to transcribed expression sites. Our results suggest that: (i) a double expressor is not a stable intermediate in expression site switching; (ii) expression sites are not independently switched on and off; and (iii) expression sites can be in a 'pre-active' silent state from which they can be readily activated.

T Cells with Two Tcrβ Chains and Reactivity to both MHC/Idiotypic Peptide and Superantigen

It is thought that Tcrβ genes are effectively allelically excluded, while Tcrα genes are not. We report here that endogenous Tcrβ genes are expressed on as much as 5–7% of CD4+T cells in 8-week-old Tcrαβ-transgenic mice. In this model, the transgenic Tcr recognizes residues 91–101 of a λ2315Ig light chain, presented on the I-Edclass II molecule. From such mice, a CD4+T cell clone was isolated which not only responded to λ2315, but also mobilized Ca2+and proliferated in response to Mls-1a. (Inadvertently we found that the C3H/Tif substrain, in contrast to C3H/HeJ, is Mls-1apositive.) The clone expressed the transgenic Tcr (Vα1 and Vβ8.2), and in addition an endogenous Vβ6 chain, conferring the Mls-1areactivity. On the population level, 1–2% of Tcr-transgenic lymph node cells displayed Vβ6, in addition to the transgenic β-chain. Such dual Tcrβ expressor cells could be preferentially expandedin vitroby first stimulating with DBA/2 spleen cells and then with λ2315-pulsed BALB/c antigen-presenting cells. In addition to demonstrating that allelic exclusion of Tcrβ-chain genes is substantial but not complete in this model, the data show that the double β-chain expressors can have two different specificities, and be signaled through both receptors, by physiological ligands. However, such dual-Tcr T cells appear to have reduced sensitivity to ligands, due to their decreased expression of each receptor. This holds true for both early (Ca2+mobilization) and late (proliferation) T cells activation parameters. Dual Tcr cells may have a role in the pathogenesis of autoimmune diseases: If naive T cells are first stimulated by (infectious) superantigen, they could later, as activated T cells, respond to self-peptide/MHC on costimulation-deficient cells and cause autoimmunity. As a corollary, dual Tcr Id-specific T cells could, once activated, directly regulate Id+B cells.

Ordered appearance of antigenic variants of African trypanosomes explained in a mathematical model based on a stochastic switch process and immune-selection against putative switch intermediates.

Antigenic variation of African trypanosomes results from the periodic activation of a single new variant cell surface glycoprotein (VSG) gene out of a repertoire of about a 1000 VSG genes. In spite of the apparently random genetic basis of the process of antigenic variation, the relapsing parasitemias are characterized by an as yet unexplained order of appearance of major VSG variants. Here we mathematically test hypotheses concerning the blood-based parasitemia. In our model the antigenic switches occur at random at the DNA level. A variable proportion of the switches has a short intermediate phase in which two different VSGs simultaneously occur on the cell surface. We show that, in a theoretical population of 230 single expressor variants in an immunocompetent or in an immunodeficient host, it is not possible to explain the ordered appearance of variants by affecting the growth coefficients of single expressors or double expressors or by affecting the antigen switch probabilities. Rather, a realistic parasitemia can be obtained if the majority of switches has a double expressor switch-intermediate phase and if the double expressors have a differential susceptibility to the immune control. This study is significant in providing a theoretical basis for the ordered appearance of variants and in explaining previously unresolved discrepancies between the rate of appearance of new variants in culture and in vivo. In addition, testable predictions as to the development of the infections, switch rate of variants, fraction of double expressors, and parasite mortality coefficients are generated.

Stable expression of two variable surface glycoproteins by cloned Trypanosoma equiperdum

African trypanosomes are thought to evade the host immune system by periodically changing their variable surface glycoprotein (VSG). VSG genes are activated by a complex process involving the duplicative transposition of silent basic copy genes to one of several expression sites. These expression-linked copies (ELCs) of the VSG genes are also subject to regulation within expression sites by as yet unknown mechanisms. It is generally assumed that trypanosomes can express only one VSG gene at a time. Nevertheless, the finding that they contain multiple VSG gene expression sites suggests that multiple expression is possible. We show here that Trypanosoma equiperdum can stably express two VSG genes in a simple axenic culture system and that both antigens are present on the cell surface. The two antigens do not co-cap or form heterodimers. Their corresponding genes show no cross-hybridization and are situated in different telomere-linked expression sites. Northern blot analysis reveals that both genes are active in the double expressors.