ALBERT

Description: Diverse perturbations to endoplasmic reticulum (ER) functions compromise the proper folding and structural maturation of secretory proteins. To study secretory pathway physiology during such “ER stress,” we employed an ER-targeted, redox-responsive, green fluorescent protein—eroGFP—that reports on ambient changes in oxidizing potential. Here we find that diverse ER stress regimes cause properly folded, ER-resident eroGFP (and other ER luminal proteins) to “reflux” back to the reducing environment of the cytosol as intact, folded proteins. By utilizing eroGFP in a comprehensive genetic screen in Saccharomyces cerevisiae, we show that ER protein reflux during ER stress requires specific chaperones and cochaperones residing in both the ER and the cytosol. Chaperone-mediated ER protein reflux does not require E3 ligase activity, and proceeds even more vigorously when these ER-associated degradation (ERAD) factors are crippled, suggesting that reflux may work in parallel with ERAD. In summary, chaperone-mediated ER protein reflux may be a conserved protein quality control process that evolved to maintain secretory pathway homeostasis during ER protein-folding stress.

Description: Although a variety of genetic alterations have been found across cancer types, the identification and functional characterization of candidate driver genetic lesions in an individual patient and their translation into clinically actionable strategies remain major hurdles. Here, we use whole genome sequencing of a prostate cancer tumor, computational analyses, and experimental validation to identify and predict novel oncogenic activity arising from a point mutation in the phosphatase and tensin homolog (PTEN) tumor suppressor protein. We demonstrate that this mutation (p.A126G) produces an enzymatic gain-of-function in PTEN, shifting its function from a phosphoinositide (PI) 3-phosphatase to a phosphoinositide (PI) 5-phosphatase. Using cellular assays, we demonstrate that this gain-of-function activity shifts cellular phosphoinositide levels, hyperactivates the PI3K/Akt cell proliferation pathway, and exhibits increased cell migration beyond canonical PTEN loss-of-function mutants. These findings suggest that mutationally modified PTEN can actively contribute to well-defined hallmarks of cancer. Lastly, we demonstrate that these effects can be substantially mitigated through chemical PI3K inhibitors. These results demonstrate a new dysfunction paradigm for PTEN cancer biology and suggest a potential framework for the translation of genomic data into actionable clinical strategies for targeted patient therapy.

Description: Members of the New Kinase Family 3 (NKF3), PEAK1/SgK269 and Pragmin/SgK223 pseudokinases, have emerged as important regulators of cell motility and cancer progression. Here, we demonstrate that C19orf35 (PEAK3), a newly identified member of the NKF3 family, is a kinase-like protein evolutionarily conserved across mammals and birds and a regulator of cell motility. In contrast to its family members, which promote cell elongation when overexpressed in cells, PEAK3 overexpression does not have an elongating effect on cell shape but instead is associated with loss of actin filaments. Through an unbiased search for PEAK3 binding partners, we identified several regulators of cell motility, including the adaptor protein CrkII. We show that by binding to CrkII, PEAK3 prevents the formation of CrkII-dependent membrane ruffling. This function of PEAK3 is reliant upon its dimerization, which is mediated through a split helical dimerization domain conserved among all NKF3 family members. Disruption of the conserved DFG motif in the PEAK3 pseudokinase domain also interferes with its ability to dimerize and subsequently bind CrkII, suggesting that the conformation of the pseudokinase domain might play an important role in PEAK3 signaling. Hence, our data identify PEAK3 as an NKF3 family member with a unique role in cell motility driven by dimerization of its pseudokinase domain.

Description: The majority of indolent B-cell lymphoma patients [pts] receive R, either as a single agent or in combination with chemotherapy. Recently, “biologic” agents (e.g. GM-CSF, Interferon-alpha, other monoclonal antibodies [mAbs]) are being combined with R in an attempt to increase its anti-tumor activity. Compared to R-chemo, these “purely” immuno-therapies avoid chemotherapy-associated non-specific toxicities while introducing unique mechanisms-of-action [MOA] against drug-resistant cells. G is a primatized anti-CD80 mAb with single-agent activity and excellent safety profile in prior psoriasis and previously treated FL trials. CD80 (i.e. B7.1) is a transmembrane glycoprotein present on the surface of various lymphoma subtypes, but also on a number of immuno-effector cells (e.g. activated macrophages, activated B-cells, dendritic cells). MOA of G includes the ability to induce ADCC on targeted malignant B-cells, but also possible immunomodulatory effects by altering cellular composition and/or cytokine profile within the tumor microenvironment. Objectives of CALGB 50402 were to determine ORR and time-to-progression [TTP] from an “extended” induction schedule of G + R (i.e. G + R weekly x 4, then every 2 months x 4) in previously untreated FL pts (WHO grades 1–3a). The base-line characteristics of 61 evaluable pts are: 61% M: 39% F; median age = 57 years (range: 22–85) with 48% of pts 〉60; 23% with elevated LDH; FLIPI: good risk = 20.3%, intermediate-risk = 42%, high-risk = 37%; histology: 44% grade 1, 46% grade 2, 10% grade 3a; 93% stage III/IV; 24% bulky (〉7 cm) disease. Therapy was very well tolerated with only 13% grade 3 adverse events. ORR is 70% (95% CI: 57% – 81.5%) and includes 44% CR/CRu, 26% PR. Greater than 10% of pts had “delayed” initial responses (i.e. 8–14 months after starting therapy) and more than 15% of pts converted from PR to CR after at least 9 months or more of therapy. Of particular interest is an apparent association of FLIPI score to ORR and CR rate: ORR (p=0.059) CR (p=0.03) FLIPI Score 0–1 11 (92%) 9 (75%) 2 20 (80%) 12 (48%) 3–5 12 (55%) 6 (27%) In further analyses, ORR was not associated with stage, gender, bulky disease, marrow involvement or age 〉 60. At a median follow-up of 2.17 years, 41 of 61 (67%) pts remain progression-free. Notably, PFS also is impacted by FLIPI score (p=0.0012): % Progressed Median PFS FLIPI Score 0–1 0 Not reached 2 24% Not reached 3–5 59% 1.62 years In conclusion, upfront extended induction G + R immunotherapy was well tolerated and achieved a current (i.e. continue to monitor for “late” responders) ORR of 70% (44% CR/ Cru). Although the FLIPI was originally developed in pts receiving chemotherapy alone, our data strongly suggest that it has applicability to a purely upfront immunotherapy (i.e. G + R) treated group of FL pts. G + R combination immunotherapy is an extremely well-tolerated and promising regimen in previously untreated FL pts with low- and intermediate-risk FLIPI status. An update, including “delayed” response rate, durability of therapeutic response, TTP, and analysis of Fc receptor polymorphisms versus response to dual monoclonal antibody therapy will be presented at the annual meeting.

Description: Background: Biologic prognostic markers in diffuse large B-cell lymphoma (DLBCL) have focused on putative cell of origin (germinal center (GC) vs. activated B-cell (ABC)), apoptosis, and proliferation. Such markers, shown to be predictive in CHOP-treated patients, are being validated with rituximab(R)-CHOP. CALGB 50103 is a phase 2 trial of dose adjusted etoposide, vincristine, doxorubicin, cyclophosphamide, prednisone, R (DA-EPOCH-R) therapy for DLBCL. We studied CD10, BCL6, MUM1, LMO2, BCL2, and Ki67 in CALGB 50103 to determine which markers had prognostic significance. Methods: Prospectively procured slides were stained with appropriate primary antibodies at the Pathology Coordinating Office of the CALGB (CD10, BCL6, MUM1, BCL2, Ki67) or the Cleveland Clinic (LMO2). Slides were scored independently in 10% increments by two pathologists, with a third review in case of disagreement of 〉20% for all stains (mean score used as final value) except Ki67, for which image analysis (IA, Aperio, Scanscope) and a visual estimate (quartiles) was used. A 30% cutoff was used for CD10, BCL6, and MUM1. 40% was used for BCL2 and 60% for Ki67. Progression-free and event-free survival (PFS, EFS) served as the endpoints. Results: Data for at least one of the markers were available for 53 of the 75 treated patients. The median age was 56 years (range 23 – 80) and the median follow-up for the 45 patients who are still alive is 4.5 years (range 3.2–6.0). The international prognostic index (IPI) was available in 51 patients and 33 had an IPI score of 2 while 18 had scores of 3 or 4. 11 patients had progressive disease and there were 14 treatment failures. 8 patie0nts have died. Table 1 shows the significant predictors of outcome. Table 1: Predictors of PFS and EFS in CALGB 50103 PFS EFS Variable No. (%) 2 yr survival prob (95% CI) 2-sided p-value 2 yr survival prob (95% CI) 2-sided p-value CI = confidence interval CD10 0.057 0.030 〈 30% 39 (76.5) 0.76 (0.59 – 0.87) 0.74 (0.58 –0.85) ≥ 30% 12 (23.5) 1.00 (---) 1.00 (---) Ki67 IA 0.028 0.045 〈 60% 32 (64.0) 0.87 (0.69 – 0.95 ) 0.84 (0.66 – 0.93) ≥ 60% 18 (36.0) 0.67 (0.40 – 0.83) 0.67 (0.40 – 0.83) IPI 0.008 0.003 2 33 (64.7) 0.91 (0.74 – 0.97) 0.87 (0.71 – 0.95) 3–4 18 (35.3) 0.65 (0.38 –0.82) 0.61 (0.35 – 0.79) Of the cell of origin markers (CD10, BCL6, MUM1, LMO2) only CD10 negativity was associated with an inferior PFS and EFS. GC vs. non-GC phenotype (as per Hans et al Blood 2004) and LMO2 were not associated with outcome. High Ki67% by IA was also associated with inferior PFS and EFS. Similar results were seen with Ki67 visual estimates (〉75% cutoff, P60% hazard ratio [HR] 7.1, P=.007; IPI 3/4 HR 7.2, P=.006) and EFS (Ki67〉60% HR 5.3, P=.007; IPI 3/4 HR 7.1, P=.002). The multivariate analysis with CD10 and IPI was not possible because there are no events in CD10+ cases. Conclusions: Lack of CD10 (suggesting non-GC origin) and high Ki67 are associated with poor outcome in DA-EPOCH-R treated patients, further validating the concept of cell of origin and proliferation as predictors of outcome in DLBCL. BCL2 remains an important predictor of outcome in patients that lack the GC marker CD10, even in rituximab treated patients.

Description: Recent studies suggest that detection of subclinical, or minimal residual disease (MRD) in apheresis products used for autografting correlates with poor disease-free survival following ASCT for mantle cell lymphoma (MCL). To validate this observation and to gain insights into the kinetics of MRD during treatment, we are performing a prospective analysis of MRD using quantitative real-time PCR (Q-PCR) in patients (pts) undergoing treatment for MCL on a CALGB study (59909). Q-PCR of sequential paired bone marrow (BM) and blood (B) samples and of apheresis products was performed using either a patient-specific immunoglobulin heavy chain (IgH) or BCL-1 gene rearrangement. All samples were analyzed in triplicate using LightCycler technology and reported as a normalized ratio of IgH or BCL-1 copy number to GAPDH copy number. The sensitivity of the assay ranged from 1 X 104–1 X 105. To date, a clonal IgH or BCL-1 gene rearrangement was detected in 36 of 41 (88%) pts entered on study. Patient-specific primers and consensus probes were used for Q-PCR monitoring of MRD following two courses of intensive induction therapy, during stem cell mobilization, and 3 and 12 months after ASCT and post-transplant immunotherapy with Rituximab (R). 27 pts have completed all protocol treatment with a median follow-up of 7 months (range: 0–28). 26 pts were evaluated for MRD following two courses of induction therapy with cyclophosphamide, methotrexate, doxorubicin, vincristine, prednisone, and R. 10 of 26 became MRD negative (−) following induction while 16 remained MRD positive (+). Following mobilization with high-dose cytarabine, etoposide and R, apheresis products from 9 of 10 MRD- pts were evaluated and all products were MRD- (1 pt not evaluable). Of the 16 pts who were MRD+ prior to mobilization therapy, MRD- apheresis products were collected in 8; 5 had MRD+ stem cell collections, and 3 were not evaluable. In total, apheresis products were evaluable in 22 of 26 pts; 17 (77%) had MRD- stem cells collected prior to ASCT. None of these MRD- pts has relapsed to date although 2 pts with MRD- products became weakly MRD+ 12 months following ASCT and R. Of the 5 pts with MRD+ apheresis collections, 4 have remained persistently MRD+ following ASCT and R; 1 has relapsed 19 months after completion of all treatment. Rising levels of MRD in BM and B samples were noted in this patient 3 and 12 months post-ASCT. Statistical comparison of MRD values in paired BM and B samples prior to, and post-ASCT demonstrated good agreement with an intraclass correlation coefficient of.814 and.777, respectively. In conclusion, our results demonstrate that prospective MRD monitoring using Q-PCR provides important insights into the kinetics of response during treatment of MCL. MRD- apheresis products were collected in the majority of pts following intensive induction and mobilization chemo-immunotherapy with R on CALGB 59909. To date, no pts with MRD- apheresis products have relapsed following ASCT. In contrast, it appears that pts with MRD+ apheresis products remain MRD+ following ASCT and R and may be more likely to relapse. Longer clinical follow-up is needed to clarify the significance of the persistence of MRD in apheresis products and following ASCT for MCL.

Description: Relapse of malignancy (RM) remains the most important cause of treatment failure following high-dose chemotherapy and autologous hematopoietic transplantation (AHCT). Allogeneic transplantation (allo-HCT) can salvage some patients who relapse or develop MDS following AHCT. However, myeloablative allo-HCT has been associated with a prohibitive transplant-related mortality (TRM) in such patients. CALGB study 100002 prospectively assessed the safety of reduced-intensity allo-HCT (RICT) in patients who failed prior AHCT. Eligibility required the development of RM or MDS 〉 6 months following AHCT for a hematologic malignancy and an available HLA-identical sibling (MSD) or 9/10 locus matched related donor, or a 10/10 matched unrelated donor. Between Dec 02 and Mar 06, 82 patients were registered from 11 CALGB centers. Patient characteristics: median age = 51 (17–70); male-63%; diagnoses - NHL 30%, HD 26%, MM 18%, MDS 16%, AML 8%, CLL 2%; donor - MSD 43%, MUD 55%, 9/10 MRD 2%. Median time between AHCT and RICT was 2.6 yrs (0.6–5.7 yrs). Preparative regimen for patients with MSD was fludarabine 30 mg/m2/d x 5 d, busulfan (i.v.) 3.2 mg/kg/d x 2 d. For patients without MSD rabbit ATG (Thymoglobulin ®) 2.5mg/kg/d x 4 d was added. PBSC 2–8 x 106 CD34+ cells/kg was used as the graft. GVHD prophylaxis consisted of tacrolimus tapering at d +90 and methotrexate 5 mg/m2 x 3 doses for MSD patients. For non-MSD patients, tacrolimus was tapered starting d +180, a fourth dose of methotrexate and MMF (d 0 to d +60) were added. Primary endpoint was TRM at 6 months. For 56 patients with 〉 6 months post-RICT follow-up (median follow-up 1.3 yrs), 6 month TRM was 8.7% (MSD patients 4.2%, non-MSD patients 9.3%). Total cumulative TRM was 14.3% (MSD 16.7%, non-MSD 12.5%). Reported cumulative rates of II–IV and III–IV acute GVHD are 28.2% and 11.8% respectively. Chimerism studies were performed centrally on peripheral blood (PB) specimens. The percentage of patients from MSD patients who achieved full (〉 90%) donor T-cell (CD3) chimerism in PB on day 30, 60, 90 and 180 post transplant are 15, 40, 47, 57 and 60% respectively. In contrast, for non-MSD patients the corresponding rates are 78, 68, 86 and 91 % respectively. Both MSD and non-MSD patients achieved full (〉90%) donor chimerism in myeloid cells by d 30 in 〉 80% of patients. This study observed a

Description: Abstract 301 Background: While whole brain radiotherapy (WBRT) has long been considered the standard consolidative therapy in primary CNS lymphoma (PCNSL), concerns regarding irreversible neurocognitive effects of brain irradiation have prompted development of dose-intensive induction and consolidative chemotherapeutic approaches, with the aim to eliminate brain irradiation. We present the updated results of the first Phase II multicenter trial, conducted by a major cooperative group within the United States, to evaluate an intensive chemotherapy-alone strategy in newly diagnosed patients with PCNSL. Methods: Induction chemotherapy consisted of methotrexate (MTX), temozolomide, rituximab (MT-R) with HD-MTX (8 gm/m2) administered every 2 weeks × 8, weekly rituximab × 6 and temozolomide (150 mg/m2) starting day +7 and continued monthly × 5. Patients who achieved a CR received intensive consolidation cytarabine 2 gm/m2 BID on days 1–4 with etoposide 40 mg/kg over 96 h (EA). Assessment of BCL6 and MYC expression by lymphoma cells was performed by immunohistochemical analysis of diagnostic specimens and scored (10% increments) by a pathologist who was blinded to clinical outcome. Results: 44 newly diagnosed, immunocompetent patients with PCNSL were treated at 12 CALGB centers between 2005 and 2009. Patient characteristics were as follows: median age was 61 yr (range 12–76), median ECOG PS was 1, 58% were IELSG risk group 2–3. 98% of tumors were large B-cell lymphoma. 66% of patients exhibited CR to induction MT-R. With median overall follow-up of 5.3 years, 21 out of 46 patients exhibited disease progression and 17 have died. There was one treatment-related death (sepsis) during consolidation. There has been no evidence for significant treatment-related neurotoxicity. The median progression-free survival (PFS) is 4.0 years and estimated PFS rates with 95% confidence limits at 1, 2, 3 and 4 years are 0.66 (0.50, 0.76), 0.59 (0.43, 0.72), 0.52 (0.36, 0.64) and 0.47 (0.32, 0.61). The probability of 2-year PFS for patients who completed the entire regimen is 0.69 (0.47, 0.94). The estimated overall survival (OS) rate with 95% confidence limits at 4 years is 0.65 (0.49,0.77). Remarkably, event-free survival (EFS) was similar in patients older and younger than 60 (p〈 0.47). While ECOG PS〉1 and high IELSG score showed a trend toward inferior EFS, the most significant clinical prognostic variable was treatment delay: those patients who started remission induction therapy more than 30 days after diagnosis exhibited shorter EFS than patients who received MT-R within a month (2-sided p-value = 0.05). There was no relationship between treatment delay and IELSG prognostic score. While high MYC expression (〉50% lymphoma nuclei) was detected in 54% of cases, MYC was not prognostic. By contrast, high BCL6 expression (≥30% of lymphoma nuclei) was detected in 59% of cases and correlated as a continuous variable with inferior progression-free survival, event-free survival and overall survival. The 2-sided p-values for these models were p=0.045, p=0.019 and p=0.045 (log-rank test). Conclusions: CALGB 50202 (Alliance) demonstrates that induction MT-R followed by EA consolidation is feasible in the multicenter setting and yields rates of PFS and OS in newly diagnosed PCNSL patients that are at least comparable to combined modality treatment involving reduced dose whole brain irradiation. The MT-R-EA regimen is well-tolerated in patients age 〉60 and has similar efficacy in this population as in younger patients. Based upon these encouraging results, a successor, intergroup, randomized phase II trial, CALGB 51101 (Alliance) has been activated that compares dose-intensive consolidation (EA) with myeloablative chemotherapy and autologous stem cell transplant in this first randomized trial in PCNSL in which neither arm involves WBRT. Our observations regarding the association of treatment delay with adverse prognosis suggest that prompt initiation of therapy for PCNSL patients may translate into improved outcomes. In this first prospective analysis of molecular biomarkers in PCNSL in the setting of a clinical trial, high BCL6 expression was found to correlate with inferior outcome. This observation raises the possibility that in future studies BCL6 could be used as a biomarker in risk-adapted therapy and supports the investigation of BCL6 antagonists in the treatment of this disease. Supported by LLS and by NCI CA13908301. Disclosures: No relevant conflicts of interest to declare.