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Dose-response curves for MEC1 and MEC2 cells treated with fludarabine.

Supplementary Table S1 Clinical characteristics of two chronic lymphocytic leukemia patient datasets; Supplementary Table S2 Clinical characteristics of two lung cancer patient datasets; Supplementary Table S3 Clinical characteristics of the ALL dataset; Supplementary Table S4 Integrated function and pathway analysis on 248 experimentally validated targets of miR-155; Supplementary Table S5 Univariate and multivariate analyses of survival with patient characteristics and miR-155 and TP53 expression as categorical and continuous variables in different patient cohorts; Supplementary Table S6 Estimate of Cox model and multivariate Cox model, as well as the HR estimated based on the model for miR-155 high and TP53 low vs. miR-155 low and TP53 high.

Purpose: The oncogenic miR-155 is upregulated in many human cancers, and its expression is increased in more aggressive and therapy-resistant tumors, but the molecular mechanisms underlying miR-155-induced therapy resistance are not fully understood. The main objectives of this study were to determine the role of miR-155 in resistance to chemotherapy and to evaluate anti-miR-155 treatment to chemosensitize tumors.Experimental Design: We performed in vitro studies on cell lines to investigate the role of miR-155 in therapy resistance. To assess the effects of miR-155 inhibition on chemoresistance, we used an in vivo orthotopic lung cancer model of athymic nude mice, which we treated with anti-miR-155 alone or in combination with chemotherapy. To analyze the association of miR-155 expression and the combination of miR-155 and TP53 expression with cancer survival, we studied 956 patients with lung cancer, chronic lymphocytic leukemia, and acute lymphoblastic leukemia.Results: We demonstrate that miR-155 induces resistance to multiple chemotherapeutic agents in vitro, and that downregulation of miR-155 successfully resensitizes tumors to chemotherapy in vivo. We show that anti-miR-155-DOPC can be considered non-toxic in vivo. We further demonstrate that miR-155 and TP53 are linked in a negative feedback mechanism and that a combination of high expression of miR-155 and low expression of TP53 is significantly associated with shorter survival in lung cancer.Conclusions: Our findings support the existence of an miR-155/TP53 feedback loop, which is involved in resistance to chemotherapy and which can be specifically targeted to overcome drug resistance, an important cause of cancer-related death. Clin Cancer Res; 23(11); 2891–904. ©2016 AACR.

Clinical correlation of miR-155 expression with survival in leukemia. Kaplan-Meier survival analysis for patients expressing high levels of miR-155 vs. low levels of miR-155 in two CLL cohorts, CLL - NEJM (A) and CLL - Italy (B), and in one ALL cohort, ALL - MDACC (C).

In vivo orthotopic lung cancer model for the role of miR-155 in chemoresistance. (A) Injection and treatment schedule for CDDP (green arrows) and anti-miR negative control (NC) or anti-miR-155 liposomal nanoparticles (red stars) for four different treatment groups: mice that were injected with A549-LVEV cells and untreated (group 1), injected with A549-LVEV cells and treated with anti-miR-NC and CDDP (group 2), injected with A549-155LV cells and treated with anti-miR-NC and CDDP (group 3), and injected with A549-155LV cells and treated with anti-miR-155 and CDDP (group 4). (B) Representative pictures of dissected mice belonging to each of the treatment groups described in panel A of this Figure. Tumor nodules are marked by dotted white circles. (C-D) Graphs of the primary tumor size (C) and aggregate mass of nodules in mediastinum (D) of the four treatment groups mentioned above. (E) In situ hybridization for miR-155 for each of the four treatment groups mentioned above. (F) Immunohistochemical analysis for Ki-67 (proliferation) and CD31 (angiogenesis), as well as the TUNEL assay (apoptosis) and TP53 immunostaining for each of the four treatment groups mentioned above.

Clinical correlation of miR-155 and TP53 expression with survival in the lung adenocarcinoma - TCGA dataset when distinguishing between TP53 wild-type and TP53 mutated samples. (A-B) Kaplan-Meier survival analysis for patients expressing high levels of miR-155 vs. low levels of miR-155 in samples that express wild-typeTP53 or harbor TP53 mutations that do not affect TP53 function (A), and in samples expressing mutated TP53 that affects TP53 function (B). (C-D) Kaplan-Meier survival analysis for patients expressing high levels of miR-155 and low levels of TP53 vs. low levels of miR-155 and high levels of TP53 in samples that express wild-type TP53 or harbor TP53 mutations that do not affect TP53 function (C), and in samples expressing mutated TP53 that affects TP53 function (D).

Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) is a distinct subtype of T-ALL with a unique immunophenotype and high treatment failure rate. The molecular genetic abnormalities and their prognostic impact in ETP-ALL patients are poorly understood.The authors performed systematic analyses of the clinicopathologic features with an emphasis on molecular genetic aspects of 32 patients with ETP-ALL.The median age was 43 years (range, 16-71). The blasts were positive for cytoplasmic CD3 and CD7 and negative for CD1a and CD8. Other markers expressed included CD34 (88%), CD33 (72%), CD117 (68%), CD13 (58%), CD5 (partial, 56%), CD2 (38%), CD10 (25%), CD56 (partial, 19%), and CD4 (6%). Cytogenetic analyses revealed a diploid karyotype in 10 patients, simple (1-2) abnormalities in 10 patients, and complex karyotype in 10 patients. Next-generation sequencing for 21 patients demonstrated that all had gene mutations (median, four mutations per patient). The most frequently mutated genes were WT1 (38%), NOTCH1 (29%), NRAS (29%), PHF6 (25%), TP53 (24%), ASXL1 (19%), FLT3 (19%), and IKZF1 (19%). All patients except one received multi-agent chemotherapy, and 22 patients underwent allogeneic stem cell transplantation. Thrombocytopenia, an abnormal karyotype, and TP53 mutation were associated with markedly shortened overall survival. Stem cell transplantation significantly improved overall survival.Patients with ETP-ALL often have high mutation burden with increased genomic instability. TP53 mutation was the only molecular prognostic marker and was associated with complex karyotype and greater than or equal to five mutations. These patients may benefit from stem cell transplantation, and recurrent gene mutations may be novel therapeutic markers.

Richter syndrome is the name given to the transformation of the most frequent type of leukemia, chronic lymphocytic leukemia, into an aggressive lymphoma. Patients with Richter syndrome have limited response to therapies and dismal survival. The underlying mechanisms of transformation are insufficiently understood and there is a major lack of knowledge regarding the roles of microRNA that have already proven to be causative for most cases of chronic lymphocytic leukemia. Here, by using four types of genomic platforms and independent sets of patients from three institutions, we identified microRNA involved in the transformation of chronic lymphocytic leukemia to Richter syndrome. The expression signature is composed of miR-21, miR-150, miR-146b and miR-181b, with confirmed targets significantly enriched in pathways involved in cancer, immunity and inflammation. In addition, we demonstrated that genomic alterations may account for microRNA deregulation in a subset of cases of Richter syndrome. Furthermore, network analysis showed that Richter transformation leads to a complete rearrangement, resulting in a highly connected microRNA network. Functionally, ectopic overexpression of miR-21 increased proliferation of malignant B cells in multiple assays, while miR-150 and miR-26a were downregulated in a chronic lymphocytic leukemia xenogeneic mouse transplantation model. Together, our results suggest that Richter transformation is associated with significant expression and genomic loci alterations of microRNA involved in both malignancy and immunity.

The cancer-risk-associated rs6983267 single nucleotide polymorphism (SNP) and the accompanying long noncoding RNA CCAT2 in the highly amplified 8q24.21 region have been implicated in cancer predisposition, although causality has not been established. Here, using allele-specific CCAT2 transgenic mice, we demonstrate that CCAT2 overexpression leads to spontaneous myeloid malignancies. We further identified that CCAT2 is overexpressed in bone marrow and peripheral blood of myelodysplastic/myeloproliferative neoplasms (MDS/MPN) patients. CCAT2 induces global deregulation of gene expression by down-regulating EZH2 in vitro and in vivo in an allele-specific manner. We also identified a novel non-APOBEC, non-ADAR, RNA editing at the SNP locus in MDS/MPN patients and CCAT2-transgenic mice. The RNA transcribed from the SNP locus in malignant hematopoietic cells have different allelic composition from the corresponding genomic DNA, a phenomenon rarely observed in normal cells. Our findings provide fundamental insights into the functional role of rs6983267 SNP and CCAT2 in myeloid malignancies.

Purpose: The oncogenic miR-155 is upregulated in many human cancers, and its expression is increased in more aggressive and therapy-resistant tumors, but the molecular mechanisms underlying miR-155-induced therapy resistance are not fully understood. The main objectives of this study were to determine the role of miR-155 in resistance to chemotherapy and to evaluate anti-miR-155 treatment to chemosensitize tumors. Experimental Design: We performed in vitro studies on cell lines to investigate the role of miR-155 in therapy resistance. To assess the effects of miR-155 inhibition on chemoresistance, we used an in vivo orthotopic lung cancer model of athymic nude mice, which we treated with anti-miR-155 alone or in combination with chemotherapy. To analyze the association of miR-155 expression and the combination of miR-155 and TP53 expression with cancer survival, we studied 956 patients with lung cancer, chronic lymphocytic leukemia, and acute lymphoblastic leukemia. Results: We demonstrate that miR-155 induces resistance to multiple chemotherapeutic agents in vitro, and that downregulation of miR-155 successfully resensitizes tumors to chemotherapy in vivo. We show that anti-miR-155-DOPC can be considered non-toxic in vivo. We further demonstrate that miR-155 and TP53 are linked in a negative feedback mechanism and that a combination of high expression of miR-155 and low expression of TP53 is significantly associated with shorter survival in lung cancer. Conclusions: Our findings support the existence of an miR-155/TP53 feedback loop, which is involved in resistance to chemotherapy and which can be specifically targeted to overcome drug resistance, an important cause of cancer-related death. Clin Cancer Res; 23(11); 2891–904. ©2016 AACR.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy, and T-ALL patients are prone to early disease relapse and suffer from poor outcomes. The PTEN, PI3K/AKT, and Notch pathways are frequently altered in T-ALL. PTEN is a tumor suppressor that inactivates the PI3K pathway. We profiled miRNAs in Pten-deficient mouse T-ALL and identified miR-26b as a potentially dysregulated gene. We validated decreased expression levels of miR-26b in mouse and human T-ALL cells. In addition, expression of exogenous miR-26b reduced proliferation and promoted apoptosis of T-ALL cells in vitro, and hindered progression of T-ALL in vivo. Furthermore, miR-26b inhibited the PI3K/AKT pathway by directly targeting PIK3CD, the gene encoding PI3Kδ, in human T-ALL cell lines. ShRNA for PIK3CD and CAL-101, a PIK3CD inhibitor, reduced the growth and increased apoptosis of T-ALL cells. Finally, we showed that PTEN induced miR-26b expression by regulating the differential expression of Ikaros isoforms that are transcriptional regulators of miR-26b. These results suggest that miR-26b functions as a tumor suppressor in the development of T-ALL. Further characterization of targets and regulators of miR-26b may be promising for the development of novel therapies.

The intricate molecular network shared between diabetes mellitus (DM) and cancer has been broadly understood. DM has been associated with several hormone-dependent malignancies, including breast, pancreatic, and colorectal cancer (CRC). Insulin resistance, hyperglycemia, and inflammation are the main pathophysiological mechanisms linking DM to cancer. Non-coding RNAs (ncRNAs), particularly microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), are widely appreciated as pervasive regulators of gene expression, governing the evolution of metabolic disorders, including DM and cancer. The ways ncRNAs affect the development of DM complicated with cancer have only started to be revealed in recent years. Insulin-like growth factor 1 receptor (IGF-1R) signaling is a master regulator of pathophysiological processes directing DM and cancer. In this review, we briefly summarize a number of well-known miRNAs and lncRNAs that regulate the IGF-1R in DM and cancer, respectively, and further discuss the potential underlying molecular pathogenesis of this disease association.

The cancer-risk-associated rs6983267 single nucleotide polymorphism (SNP) and the accompanying long noncoding RNA CCAT2 in the highly amplified 8q24.21 region have been implicated in cancer predisposition, although causality has not been established. Here, using allele-specific CCAT2 transgenic mice, we demonstrate that CCAT2 overexpression leads to spontaneous myeloid malignancies. We further identified that CCAT2 is overexpressed in bone marrow and peripheral blood of myelodysplastic/myeloproliferative neoplasms (MDS/MPN) patients. CCAT2 induces global deregulation of gene expression by down-regulating EZH2 in vitro and in vivo in an allele-specific manner. We also identified a novel non-APOBEC, non-ADAR, RNA editing at the SNP locus in MDS/MPN patients and CCAT2-transgenic mice. The RNA transcribed from the SNP locus in malignant hematopoietic cells have different allelic composition from the corresponding genomic DNA, a phenomenon rarely observed in normal cells. Our findings provide fundamental insights into the functional role of rs6983267 SNP and CCAT2 in myeloid malignancies.

The role of non-protein coding RNAs (ncRNAs), microRNAs (miRNAs) in particular, as fine-tuners of both pathological and physiological processes is no longer a matter of debate. With the recent discovery of miRNAs in a wide variety of body fluids and considering them as tools employed in horizontal gene transfer between cells, a new horizon opens in the field of diagnosis and therapeutics. Circulating miRNAs not only enable the communication among cells, but also provide insight into the pathological and physiological state of the originating cells. In this review we summarize the recent advances made in this field, arguing for compelling translation of miRNAs into clinical practice. Moreover, we provide overview of their characteristics and how they impact the evolution of tumor microenvironment and cell-to-cell communication, advancing the idea that miRNAs may function as hormones.

Familial adenomatous polyposis (FAP) can be considered as a condition of the whole body as extracolonic features derived from all the three embryonic lineages are recorded with varying frequency in addition to the presence of multiple adenomas in the large intestine. Here, we describe two unrelated cases of FAP with unusual extracolonic phenotypes, namely several abnormalities of mesodermal origin strongly resembling Marfan syndrome (MFS) or a Marfan-like habitus. Conventional cytogenetic and FISH analysis did not reveal any gross chromosomal rearrangement on the long arm of chromosome 5 where the APC and FBN2 genes were located. However, in case 2 the FAP-causing mutation in the APC gene was found in the donor splice site of exon 4 and was shown to result in a frameshift and a premature termination codon. We propose that such connective tissue abnormalities may result from germline APC mutations in combination with specific genetic and/or environmental modifying factors.

We evaluated long-term outcomes of 60 patients with chronic lymphocytic leukemia treated with an initial therapy of lenalidomide. At a median follow-up of 4 years, time-to-treatment failure has not been reached and overall survival is 82%. Thirty-five (58%) patients had a response lasting >36 months (long-term responders [LTRs]). Best LTR responses consisted of 25 (71%) complete remissions and 10 (29%) partial remissions. In addition to clinical responses, an increase in IgA, IgG, and IgM levels of >50% from baseline was reported in 61%, 45%, and 42% of LTRs. Normalization in the percentage of CD4+ and CD8+ cells and T-cell numbers was observed in 48%, 71% and 99% of LTRs. Compared with other patients in the study, LTRs had lower baseline plasma levels of β-2-microglobulin, were more likely to have trisomy 12, and less likely to have deletion 17p. This trial was registered at http://clinicaltrials.gov as # NCT00535873.

Noncoding RNAs play a pivotal role in the pathogenesis of chronic lymphocytic leukemia (CLL). We hypothesized that microRNAs (miRs) are involved in the transition from monoclonal B-cell lymphocytosis (MBL) to CLL and tested miR-15a/16-1 cluster, miR-21, and miR-155 expression in purified B cells of normal individuals, individuals with MBL, and patients with CLL. When we analyzed 224 samples from 2 independent training and validation cohorts, we found that miR-155 was overexpressed in B cells from individuals with MBL, and even more so in B cells from patients with CLL, when compared with B cells from normal individuals. Furthermore, we were able to identify miR-155 in circulating microvesicles from both individuals with MBL and patients with CLL. Next, to examine the prognostic role of miR-155, we measured its expression level in plasma samples collected before treatment initiation in 228 patients with CLL. We found significantly higher miR-155 expression levels in patients who failed to achieve a complete response compared with those who experienced complete response. Our findings support the use of cellular and plasma levels of miR-155 as biomarkers for the risk of progression in individuals with MBL, as well as to identify patients with CLL who may not respond well to therapy.

We present the case of a 72-year-old man with atrial fibrillation who developed an embolic occlusion of the superior mesenteric artery. He was successfully treated with local fibrinolysis using streptokinase associated with angioplasty. Such local treatments without the need of surgery are very rarely reported in the literature because of the great difficulty in selecting patients without intestinal necrosis, but represent an important option in the algorithm for the management of acute mesenteric ischemia. The patient was in good condition without recurrent embolism during the six months follow-up.

Abstract 3932 The best initial treatment for elderly patients (pts) with CLL is evolving and is a field of active investigation. We conducted a phase II study evaluating the activity of lenalidomide as initial therapy for elderly pts with CLL. This treatment was associated with an overall response rate of 65% and an overall survival of 88% at 2 years (Badoux, Blood 2011). Because response duration is a relevant endpoint, we performed an analysis looking at long-lasting responses to lenalidomide in pts on this trial. Pts with a response lasting 36 months or longer were defined as “long-term responders” (LTRs) and are the focus of our report. Clinical characteristics, prognostic factors, serum immunoglobulin levels, circulating T cell numbers (up to 36 months) and plasma cytokine levels of LTRs were compared with the rest of the study population using non-parametric and Chi-square tests. Differences were considered to be significant if p was < 0.05. Thirty-one of the 60 pts (52%) are LTRs. Best responses among LTRs consisted of 29 complete remissions (CR), including 4 pts with MRD-negative CR, and 2 partial remissions. Median time to failure (TTF) has not been reached for LTRs, after a median follow up of 47 (37–53) months. The median daily dose of lenalidomide at last follow-up in these pts was 5 mg (2.5–10). Twenty-two LTRs are still on therapy and nine have discontinued lenalidomide. Reasons for treatment discontinuation were: toxicity in 6 pts (deep venous thrombosis after 41 months in 1 pt, moderate neuropathy after 30 and 39 months in 2 pts, persistent fatigue after 23 months in 1 pt, moderate weight loss after 5 months in 1 pt, immune thrombocytopenia after 11 months in1 pt), infectious complications occurred in 1 pt (sepsis, after 12 months), second malignancy (invasive squamous cell carcinoma of the skin after 26 months) in 1 pt and change of institution in 1 pt. LTRs experienced neutropenia during the first 12 months of therapy that later resolved in 83% of pts (Fig 1A); additionally a recovery in hemoglobin and platelets compared to baseline values was also seen in 100 and 77% of LTRs, respectively (Fig 1B-C). We also observed an increase in the percentage of circulating T cells (CD3+) (Fig 1D) and an increase in plasma levels of immunoglobulins A, G and M values in these pts (Fig 2). Lenalidomide-related late toxicities observed in LTRs consisted of G 1–2 diarrhea in 2 pts and G1 peripheral neuropathy in 3 pts. One patient developed several skin lesions shown to be in situ squamous and basal cell carcinomas. When we compared pre-treatment clinical characteristics of LTRs and the other pts on study, we observed that LTRs had lower baseline beta-2-microglobulin (median values: 4 vs 4.8 mg/L; p=0.02) and were less likely to have a deletion 11q (5 vs 9; p=0.02). Furthermore, baseline plasma levels of IL6, IL8, IFNγ and MIP1α were significantly lower in the LTRs (p=0.002, 0.025, 0.044 and 0.001, respectively). In conclusion, lenalidomide as initial therapy of elderly pts with CLL induced responses lasting more than 3 years in 53% of pts. Compared to the pts with shorter response duration, LTRs were more likely to have lower baseline levels of beta2-microglobulin, IL6, IL8, IFNγ and MIP1α and intermediate or favorable cytogenetic abnormalities. Fig 1. Fig 1. Fig 2. Fig 2. Disclosures: Keating: Celgene Corporation: Consultancy. Wierda:Celgene Corporation: Consultancy. O'Brien:Celgene Corporation: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Ferrajoli:Celgene Corporation: Honoraria, Research Funding.

Abstract 1788 Patients with relapsed CLL have limited effective treatment options, and new therapies are needed. We report the results of a phase II study which investigated the efficacy and tolerability of a combination regimen with ofatumumab and lenalidomide in patients (pts) with relapsed CLL. These agents were evaluated as a combination regimen for pts with relapsed CLL based on their documented single-agent efficacy, distinct and potentially complimentary mechanisms of action and non-overlapping toxicities. Furthermore, the combination of rituximab and lenalidomide was active in relapsed CLL (Badoux et al. 2010). Pts with symptomatic disease who met treatment indications were eligible. Other inclusion criteria required prior treatment with purine analogs, an ECOG PS score of 0–2, and adequate organ function (creatinine clearance > 30ml/min, total bilirubin < to 2 mg/dl, ALT < 2 × ULN). Pts with any neutrophil count were eligible. Pts were excluded for platelets < 30,000 mm3, positivity for HIV, active hepatitis B or C or recent history of tuberculosis. Pts received ofatumumab IV weekly for four weeks (300mg week 1; 1,000 mg weeks 2 to 4), monthly during months 2–6, and every other month during months 7–24. Lenalidomide 10 mg PO daily was started on day 9 and continued daily for a maximum of 24 months. Allopurinol 300mg PO daily was given during the first two weeks of cycle 1. No pts received antibiotic or DVT prophylaxis. The use of growth factors was according to standard guidelines. Responses were assessed after 3, 6, 12, 18 and 24 months. Between January 2010 and January 2011, 36 pts were enrolled and 34 pts are evaluable (one pt withdrew consent prior to treatment and one was excluded because of concomitant MDS). The median age of the pts was 62 yrs (34–82). Twenty pts (59%) had Rai III-IV disease. The median beta-2M level at start of therapy was 4.4 mg/dL (1.7-16.5). The median number of prior treatments was 2 (1–8). All pts had received prior chemoimmunotherapy with fludarabine, cyclophosphamide and rituximab (FCR). Twelve pts (35%) were fludarabine-refractory. Twenty-two pts (65%) had unmutated IgHV genes, 9 (26%) del(17p) and 4 (12%) del(11q). Responses were evaluated according to the 2008 NCI-WG criteria: 5 pts (15%) achieved CR (including 1 CRi) and 17 pts (50%) PR, for an ORR of 65%. Of the 5 CR pts, 1 was MRD-negative by flow cytometry. Two of the PR pts achieved marrow CR and only had persistent lymphadenopathy. Of the 22 responders, 13 are continuing on therapy with ongoing response (median time on therapy 14 months, range 8–20 months). Despite ongoing response, 7 pts discontinued therapy due to: transition to HSCT (3 pts; after 3, 6 and 6 months, respectively), pulmonary embolism (1 pt; after 4 months), recurrent zoster infection (1 pt; after 17 months of therapy) and physician choice (2 pts; after 9 and 16 months of therapy). Two pts discontinued therapy because of loss of response after 12 and 18 months of treatment, respectively. Thirty-one pts (91%) are alive. There have been no deaths on therapy. Three deaths occurred after discontinuation of therapy due to refractory disease (1), following HSCT (1) and due to progressive disease and adenocarcinoma of the lung (1). The most common grade 3–4 treatment-related hematological adverse events included neutropenia (15 pts, 44%), thrombocytopenia (3 pts, 9%) and anemia (1 pt, 3%). Thrombotic events occurred in 2 pts (6%). One pt experienced grade 3 pulmonary embolism and another patient grade 2 superficial vein thrombosis. In both cases with thrombotic event, ESAs were concomitantly administered. One pt (3%) experience a grade 3 infusion reaction during the first ofatumumab administration. Eight grade 3 infectious episodes occurred: pneumonia (3), neutropenic fever (2), parotitis (1), LE cellulitis (1) and CNS toxoplasmosis based on radiological findings (1). Intermittent grade 1–2 diarrhea was reported by 8 pts (24%). Grade 1–2 lenalidomide-associated tumor flare reaction was seen in 8 pts (24%). The median tolerated daily dose of lenalidomide was 5 mg/day (2.5-10 mg). In conclusion, the combination of ofatumumab and lenalidomide is well tolerated. Neutropenia was the most common toxicity. This combination induced responses in 65% of pts with relapsed CLL, all of whom had received prior chemoimmunotherapy. Responses are durable and ongoing in some pts. Disclosures: Ferrajoli: GlaxoSmithKline: Research Funding; Celgene: Research Funding. O'Brien:GlaxoSmithKline: Consultancy; Celgene: Consultancy. Wierda:GlaxoSmithKline: Research Funding.

Abstract 3907 After treatment with purine analog-based combination chemoimmunotherapy, 40% to 60% of patients with CLL have residual disease. As the extent of the residual disease often correlates with time to progression, eradication of residual disease may improve the duration of remission. Although alemtuzumab has been used as consolidation therapy with improvement of responses in 56% of the patients (pts) (Montillo et al, 2006), its use as been limited by concerns of immunosuppression. Lenalidomide is an oral agent with activity in pts with CLL. Lenalidomide's easy oral administration and its immunomodulatory properties make it an interesting agent to be evaluated as consolidation therapy. We, therefore, designed a phase II study to evaluate the efficacy and toxicity of lenalidomide for treatment of residual disease after chemotherapy. Pts with stable PR (at two time assessments at least 3 months apart), nodular PR (nPR), or CR with evidence of disease on immunophenotyping were eligible. Pts were required to enroll between 3 and 9 months after completion of combination chemotherapy. Pts received lenalidomide 10mg daily for 12 months (the first 12 pts in this trial received lenalidomide for 3 months, and the study was then amended to allow up to 12 months of therapy). Early discontinuation of treatment was allowed if pts achieved negativity for minimal residual disease (MRD) before 12 months. Responses were evaluated at 4 months and 12 months according to 1996 NCI-WG criteria. Bone marrow biopsy and BM immunophenotyping by multicolor flow cytometry (MFC) with quantification of residual disease were performed at the time of response assessment. The MFC assay used has a sensitivity of 0.02% (20 aberrant cells/100,000 total cells analyzed). Twenty-six pts have been enrolled in this study, and 21 are evaluable for response. Three pts discontinued treatment within the first 28 days (after 2, 7, and 21 days of therapy) and 2 pts are too early for response assessment. The median age is 62 years (range, 41–76), median number of prior treatment was 2 (range, 1–12). Six pts entered the study in CR, 8 pts had nPR and 12 pts were in PR. The outcome of lenalidomide consolidation and duration of therapy are summarized in the table.Pt #Latest chemotherapyResponse at start of lenalidomideDuration of therapyResponse at end of lenalidomide1FCRCR2 daysNot evaluable2OFARCR49 daysProgression3FCRCR21 daysNot evaluable4R-hyperCVADCR12 monthsCR, MRD +5FCRCR5 monthsCR, MRD +6FBRCR10 days +Too early7FCMRnPR3 monthsCR, MRD +8FCRnPR3 monthsnPR9R-CHOPnPR3 monthsCR, MRD −10CFARnPR3 monthsCR, MRD −11R-hyperCVADnPR12 monthsnPR12FCRnPR3 monthsnPR13FCR bevacizumabnPR12 monthsnPR14FCR bevacizumabnPR4 monthsnPR15OFARPR3 monthsCR, MRD −16CFARPR3 monthsPR17PCRPR3 monthsPR18FCRPR3 monthsnPR19FCRPR3 monthsPR20FCRPR6 monthsnPR21FCR bevacizumabPR9 months +CR, MRD +22FCRPR9 months +PR23FCRPR8 months +PR24FCRPR7 daysNot evaluable25FCR bevacizumabPR7 months +PR26FCRPR3 months +Too early For the 6 pts in CR at study initiation: 2 pts had no changes in MRD status, 1 pt had disease progression, 2 pts are not evaluable due to early discontinuation and 1 pt has not had enough time on study. For the 8 pts with an nPR: 3 improved response to CR (2 of them MRD negative) and 5 pts had stable nPR. For the 12 pts with a PR: 2 pts improved to a CR (1 of them MRD negative), 2 pts to a nPR, 5 pts remained in PR, 1 pt is not evaluable due to early discontinuation and 1 pt has not had enough time on study. At a median follow up of 23 months, the median time to progression has not been reached in the 7 pts that improved their response as a result of th, and 5 pts remain in remission between 18 and 35 months after therapy. The most common lenalidomide-related adverse event was myelosuppression: grade 3–4 neutropenia (8 pts, 31%) and grade 3–4 thrombocytopenia (2 pts, 8%). Infections occurred in 5 patients: 3 pneumonia (1 RSV, 1 Pseudomonas and 1 unknown pathogen), 1 vaginal Herpes, 1 sinusitis. One patient experienced a grade 4 PE. Other common grade 1–2 toxicities considered lenalidomide-related were fatigue (8 pts, 31%), rash (8 pts, 31%), constipation (4 pts, 15%), diarrhea (4 pts, 15%), cough (3 pts, 8%) and body aches (3 pts, 8%). Based on our early experience, lenalidomide consolidation after chemotherapy can further improve responses in 27% of patients with CLL. Elimination of MRD was seen in 12% of patients treated. Enrollment to this study is ongoing. Disclosures: Keating: Celgene: Consultancy. O'Brien:Celgene: Consultancy. Ferrajoli:Celgene: Research Funding.

Abstract 4633 Background: Golgins are proteins of the Golgi complex. Several Golgins have been implicated in apoptosis. Expression of Golgin-84, a Golgin protein, is altered in apoptotic WEHI-231, a B-cell lymphoma line, suggesting that Golgin-84 may play a role in lymphoid tumorigenesis. Here, we aimed to determine the expression levels of Golgin-84 in human primary non-Hodgkin lymphomas and plasma cell myeloma. Design: Golgin-84 expression was investigated in non-Hodgkin lymphoma cell lines by using Western blot analysis and polyclonal antibodies. Using immunohistochemical stains, Western blotting analysis and Q-PCR, Golgin-84 expression was assessed in 5 reactive lymph nodes, 149 cases of primary non-Hodgkin lymphoma and 28 cases of primary plasma cell myeloma. Results: Immunohistochemical stains, Western blotting analysis and Q-PCR on 5 reactive lymph nodes demonstrated that Golgin-84 was expressed at low levels in lymphoid cells of germinal centers, mantle cells, marginal zones, and interfollicular areas. Golgin-84 was variably expressed in non-Hodgkin lymphoma cell lines tested, with the highest levels in cells from high-grade tumors (e.g. anaplastic large cell lymphoma; ALCL, Diffuse large B-cell lymphoma (DLBCL), ALCL and peripheral T-cell lymphoma unspecified (PTCL)) and the lowest levels in mantle cell lymphoma (MCL) cells. DLBCL, ALCL and PTCL frequently showed high expression of Golgin-84. Most lymphoplasmacytic lymphomas (LPL) and plasma cell myeloma (PCM) expressed high levels of Golgin-84. Expression levels of Golgin-84 were lower in MCL and low-grade B-cell non-Hodgkin lymphomas, including chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), follicular lymphoma (FL), and marginal zone lymphoma (MZL). Conclusions: Golgin-84 expression levels are low in lymphoid cells of normal lymph nodes. Most (>90%) cases of LPL and PCM, and at least half of cases of DLBCL, ALCL and PTCL express high levels of Golgin-84. These findings suggest that Golgin-84 may be involved in tumorigenesis or lymphoma progression, particularly in neoplasms with plasmacytic differentiation. Disclosures: No relevant conflicts of interest to declare.