No abstract

Clinical trials and correlative laboratory research are increasingly reliant upon archived paraffinembedded samples. Therefore, the proper processing of biological samples is an important step to sample preservation and for downstream analyses like the detection of a wide variety of targets including micro RNA, DNA and proteins. This paper analyzed the question whether routine fixation of cells and tissues in 10% buffered formalin is optimal for in situ and solution phase analyses by comparing this fixative to a variety of cross linking and alcohol (denaturing) fixatives. We examined the ability of nine commonly used fixative regimens to preserve cell morphology and DNA/RNA/protein quality for these applications. Epstein-Barr virus (EBV) and Bovine Papillomavirus (BPV)-infected tissues and cells were used as our model systems. Our evaluation showed that the optimal fixative in cell preparations for molecular hybridization techniques was "gentle" fixative with a cross-linker such as paraformaldehyde or a short incubation in 10% buffered formalin. The optimal fixatives for tissue were either paraformaldehyde or low concentration of formalin (5% of formalin). Methanol was the best of the non cross-linking fixatives for in situ hybridization and immunohistochemistry. For PCR-based detection of DNA or RNA, some denaturing fixatives like acetone and methanol as well as "gentle" cross-linking fixatives like paraformaldehyde out-performed other fixatives. Long term fixation was not proposed for DNA/RNA-based assays. The typical long-term fixation of cells and tissues in 10% buffered formalin is not optimal for combined analyses by in situ hybridization, immunohistochemistry, or -if one does not have unfixed tissues -solution phase PCR. Rather, we recommend short term less intense cross linking fixation if one wishes to use the same cells/tissue for in situ hybridization, immunohistochemistry, and solution phase PCR.

2788 Preclinical studies conducted at our institution (Alinari et al. Blood Abstract 1694, 2009) demonstrated superior efficacy of milatuzumab (Immunomedics, Inc.), a humanized anti-CD74 antibody, in combination with rituximab in mantle cell lymphoma (MCL) cell lines, MCL patient (pt) primary tumor cells, and an in vivo preclinical model of human MCL, compared to either agent alone. Veltuzumab (Immunomedics, Inc.), a novel humanized anti-CD20 antibody, has been reported to have several advantages over rituximab including slower off-rates, shorter infusion times, higher potency, and improved therapeutic responses in animal models. Phase II clinical testing of veltuzumab demonstrated single agent activity in pts with relapsed and refractory non-Hodgkin's lymphoma (NHL). In vitro, veltuzumab combined with milatuzumab also resulted in enhanced apoptosis compared to either agent alone, similar to that observed with rituximab and milatuzumab (Fig 1). As a result of the anti-tumor activity observed in vitro with combined veltuzumab and milatuzumab, we initiated a phase I/II trial in pts with relapsed or refractory B-cell NHL after at least 1 prior therapy to determine the safety, tolerability, and overall response rate with this combination. Patients received veltuzumab 200 at mg/m2 weekly combined with escalating doses of milatuzumab at 8, 16, and 20 mg/kg twice per wk of wks 1–4, 12, 20, 28, and 36. All pts received pre-medication with acetominophen, diphenhydramine, and famotidine prior to each veltuzumab dose and acetominophen, diphenhydramine, and hydrocortisone 50 mg before and after each milatuzumab dose. Dose limiting toxicity was defined during weeks 1–4. Six pts with grade 2 (n=2) or 3 (n=4) follicular NHL, have completed at least 4 weeks of combined veltuzumab and milatuzumab. Median age was 63.5 years (range 44–81), and pts received a median of 3.5 prior therapies (range 3 – 5), including 2 pts with prior autologous stem cell transplant. Three of 6 patients were refractory to rituximab, defined as having less than a partial response to the last rituximab-containing regimen. Dose escalation has reached 16 mg/kg milatuzumab, and no dose limiting toxicities have been observed to date. However, 3 of 6 pts experienced grade 3 infusion reactions during or immediately following the milatuzumab infusion; 1 pt treated with 8 mg/kg milatuzumab during weeks 1 and 12, and 2 pts receiving 16 mg/kg during weeks 3 and 12. Grade 3 infusion reactions consisted of fever, rigors, nausea, vomiting, diarrhea, and in 1 case a diffuse macular rash. Grade 3–4 hematologic toxicity occurred in only 2 pts consisting of grade 4 lymphopenia, and no infections have been observed. The most frequently observed grade 1–2 toxicities were fatigue, transient hyperglycemia, dyspnea, hypoalbuminemia, and thrombocytopenia. Human anti-veltuzumab and anti-milatuzumab antibodies, collected in all 6 pts pretreatment and day 1 of weeks 4 and 12, have not been detected in any pt. Plasma cytokine levels of IL-10, IL-12, TNF-α and INF-γ were checked pre- and post- veltuzumab infusion on day 1 and pre- and post-milatuzumab infusion on day 2. While elevations in cytokine levels were observed, there was no correlation with infusion reactions or response. In the first cohort, one pt achieved a PR maintained at week 20, 1 pt experienced stable disease at week 10, and 1 pt developed progressive disease at week 20. In the second cohort, two pts achieved a PR at week 10, and 1 pt had stable disease at week 10. Two of 3 responding pts were rituximab-refractory. Due to the observed infusion reactions with milatuzumab, the protocol has been amended to include additional premedication with intravenous antihistamine, and dexamethasone 20 mg pre-milatuzumab and 10 mg post-milatuzumab. The schedule of treatment was also modified so that the antibodies are no longer administered on the same day. Dose escalation will continue and updated results will be presented. The first 2 cohorts will be expanded to 6 pts to determine if the modifications will limit the infusion reactions and permit prolonged dosing in responding pts. In summary, 3 of 6 pts with relapsed or refractory NHL treated to date including heavily pretreated and rituximab-refractory pts, responded to combination therapy, achieving a PR. The primary observed toxicity has been infusion reactions due to milatuzumab. Disclosures: Christian: Immunomedics, Inc.: Research Funding. Off Label Use: The use of the monoclonal antibodies veltuzumab and milatuzumab is experimental in the treatment of non-Hodgkin's lymphoma. Benson:Immunomedics, Inc: Research Funding. Jones:Immunomedics, Inc.: Research Funding. Wegener:Immunomedics, Inc.: Employment, shareholders. Goldenberg:Immunomedics, Inc.: Employment, shareholders. Baiocchi:Immunomedics, Inc.: Research Funding. Blum:Immunomedics, Inc.: Research Funding.

Patients with HIV-associated lymphocyte-depleted Hodgkin lymphoma (HIV-HL) often present with advanced, extranodal disease and aggressive clinical features, limiting definitive therapeutic intervention. Here we report two patients with HIV-HL who presented with multi-organ dysfunction as an initial manifestation of their malignancy. Both were initially treated with brentuximab vedotin (BV), which led only to a temporary partial response, highlighting the challenges of treatment. One patient was eventually started on nivolumab and responded very well to the immune checkpoint inhibitor. To our knowledge, this is the first case to describe successful use of nivolumab in a patient with relapsed lymphocyte-depleted HIV-HL. Prompt recognition of multi-organ dysfunction as an initial presentation of lymphocyte-depleted HIV-HL is essential to ensure rapid provision of therapy. While use of BV remains a reasonable option, earlier introduction of immunotherapy in the treatment of HL may provide an additional option in critically ill patients with lymphocyte-depleted HIV-HL.

595FN2 Introduction: Mantle cell lymphoma (MCL) is an incurable B-cell non-Hodgkin lymphoma characterized by aberrant genetic (t(11;14)(q13;q32)) and epigenetic (DNA hypermethylation) dysregulation. Chromatin remodeling complexes and associated co-repressors such as histone deacetylases (HDAC), DNA methyltransferases (DNMT) and protein arginine methyltransferase 5 (PRMT5), are involved in silencing tumor suppressor and regulatory gene expression and may contribute to B-cell transformation. PRMT5 silences the transcription of key regulatory genes by symmetric di-methylation (S2Me) of arginine (R) residues on histone proteins (H4R3 and H3R8). We have previously identified PRMT5 over expression to be relevant to MCL pathogenesis and shown it to work concertedly with HDAC2, methyl-CpG binding domain protein 2 (MBD2) and DNMT3a to silence genes with anti-cancer and immune modulatory activities. siRNA-mediated knockdown of PRMT5 in MCL cell lines leads to growth arrest and apoptosis, thus, we explored methods to inhibit PRMT5 activity as a novel experimental therapeutic strategy for this disease. Methods and Results: A rational design of small molecule compounds to inhibit PRMT5 activity led us to construct an in silico model of the human PRMT5 catalytic domain based on available homologous crystal structures from Protein Data Bank (MODELLER9v1 software). We screened a library of 10,000 compounds and eight small molecules were identified for biological investigation based on binding energy in the PRMT5 catalytic site. Enzyme inhibition assays using purified PRMT1 (type I PRMT) and PRMT5 (type II PRMT) showed that two compounds (BLL1 and BLL3) were capable of selectively inhibiting PRMT5 and not PRMT1 activity (p<0.0001). PRMT methylation assays were also performed with SWI/SNF complexes containing PRMT5, PRMT7 (type II) or PRMT4 (type I) and both BLL1 and BLL3 demonstrated selective PRMT5 inhibition. Both drugs interfered with maintenance of S2Me-H4R3 and S2Me-H3R8 in MCL cell lines by western blot and confocal microscopy. Dose titration experiments with BLL1 (10uM - 100uM) showed a dose-dependent response of inhibition of cellular proliferation, induction of cell cycle arrest, and promotion of caspase-independent cell death in 7 MCL cell lines. BLL1 treatment of MCL cells resulted in down modulation of cyclin D1 and Mcl1, critical molecules involved in the pathogenesis of MCL. The loss of cyclin D1 and Mcl1 expression occurred as early as 1 hour after treatment with BLL1 (50uM). PRMT5 associates with the co-repressors HDAC2, MBD2 and DNMT3a on target gene promoters, thus we next evaluated the effect of BLL1 on transcriptional repression of known target anti cancer genes. The association with other co-repressors provided rationale for examining PRMT5 inhibition alone and in combination with agents that target epigenetic processes. Combination treatment of MCL cells with subtoxic doses of BLL1 (25uM), hypomethylating agent (5-azacitidine, 500nM) and HDAC inhibitor (TSA 75nM) showed synergistic induction of cell death and loss of S2Me-H4R3. Analysis of the ST7 tumor suppressor, a target repressed by PRMT5, showed mRNA levels to increase 5–7-fold following treatment with BLL1. Preclinical in vivo studies have shown favorable toxicity and pharmacokinetic profiles for both BLL1 and BLL3. In vivo evaluation of BLL1 in a preclinical, xenograft model of human MCL are currently in progress. Primary tumors of 46 patients with MCL (common, blastoid or pleomorphic histology) demonstrated abundant PRMT5 expression in both cytoplasmic and nuclear compartments (87% PRMT5 pos). Conclusions: We have successfully developed a new class of drug to selectively target PRMT5 enzymatic activity. PRMT5 over expression is linked with post translational modification of both histone and non histone proteins that contribute to key oncogenic pathways in MCL. Inhibition of type II PRMT enzymes reverses transcriptional repression of anti cancer genes and restores important regulatory cellular checkpoints of cell growth and survival. We are currently developing drugs with improved selectivity and potency. The anti tumor activity of this novel class of drug and PRMT5 expression profiles seen in MCL primary tumor specimens, supports further exploration of targeting this pathway in hematologic malignancies. Disclosures: No relevant conflicts of interest to declare.