ALBERT

Description: Abstract 2716 Background Btk is a tyrosine kinase involved in B cell receptor (BCR) signal transduction. Recent studies indicate that chronic active BCR signaling is a pathogenic mechanism in ABC DLBCL that engages the classical NF-κB pathway. Mutations in the BCR pathway (CD79A/B and CARD11) and toll like receptor (TLR) pathway (MYD88) lead to constitutive NF-κB activation in ABC DLBCL. PCI-32765 kills ABC DLBCL cell lines with constitutive BCR signaling but has no effect on ABC and GBC DLBCL cell lines that do not rely on constitutive BCR signaling. PCI-32765 is an oral, well-tolerated and irreversible inhibitor of Btk. Study Design Patients with relapsed/refractory ABC DLBCL received PCI-32765 at a fixed dose of 560 mg po once daily × 35 days (1 cycle). Patients underwent CT and FDG-PET scanning pre-treatment and every 2 cycles. Where possible, pre-treatment and 48-hour post-treatment tumor biopsies were performed for gene expression profiling (GEP) and mutational analysis of CD79A/B, CARD11 and MYD88. Results Eight of 15 planned patients are enrolled. Characteristics include median (range) age 54 (40–79); LDH 〉 normal limits (63%); any extranodal site (75%) and stage 4 disease (63%). IPI distribution was 0–2 (25%) and 3–5 (75%). Patients received a median (range) of 3 (1–6) prior chemotherapy regimens. Best response by IWG criteria include CR: 2 (25%) for 11+ and 5 months; SD (stable disease) 3 (37%) for 4, 2 and 2 months; and PD (progressive disease) 3 (38%). One patient who was primary refractory achieved SD with PCI-32765, associated with a 25% tumor reduction, and is currently in CR following allogeneic BMT. PCI-32765 was well-tolerated without significant side effects. No patients discontinued PCI-32765 due to AE. Grade 〉3 AEs were reported in 4 patients, none were considered related to PCI-32765. One death has occurred on study, in a patient who received subsequent therapy following progression on PCI-32765. Toxicities that are possibly related to PCI-32765 include diarrhea (grade 1) in 2 patients, nausea (grade 1) in 2 patients and fatigue (grades 1–2) in 4 patients. CD79B mutations were uncovered in two patients, the patient with SD who achieved a 25% tumor response and one patient who achieved CR. Of note, the other patient who achieved CR did not have the CD79B mutation, suggesting that chronic active BCR signaling may occur in the absence of this mutation. None of the patients had MYD88 or CARD11 mutations. Comparison of the pre-treatment and on-treatment biopsy samples by gene expression profiling was completed on 5 patients (1 CR, 4 SD/PD). Gene expression signatures reflecting tumor infiltrating immune cells, including CD8+ T cells and macrophages, were diminished by PCI-32765 treatment in the one patient in CR and the one patient in SD who achieved a 25% tumor reduction, but not in the others. In these same two cases, a gene expression signature of interferon signaling was reduced upon PCI-32765 treatment, associated with a concomitant decrease in interferon gamma mRNA levels, again suggesting the loss of activated T cells in the responding tumors. One hypothesis to explain this observation would be cytokine modulation since chronic active BCR signaling promotes synthesis and secretion of IL-10 and multiple chemokines, including CXCL13, CXCL9 and CCL3, which were all down-modulated in the responding tumors. Conclusions The Btk inhibitor PCI-32765 has clinical activity in relapsed/refractory ABC DLBCL and modulates chronic active BCR signaling in responders. Thus, chronic active BCR signaling is a tractable therapeutic target in ABC DLBCL. Disclosures: Buggy: Pharmacyclics, Inc.: Employment. Hedrick:Pharmacyclics: Employment, Equity Ownership.

Description: Background: Currently, diagnosis of aggressive B cell non-Hodgkin lymphomas (agg-B-NHL) uses a varying combination of morphology, immunophenotyping, cytogenetics, and/or other molecular techniques resulting in a fragmented, confusing diagnostic system. We sought to develop a multi-analyte gene expression signature assay that could consolidate the diagnostic process into a single platform to improve standardization and accuracy. Methods: We used formalin-fixed, paraffin-embedded tissue biopsies, qualified by an expert Hematopathology review panel, tumor content of ≥60%, and confirmed B cell immunophenotype. Diagnostic categories included diffuse large B cell lymphoma (DLBCL) including the activated B cell-like (ABC), germinal center B cell-like (GCB) subtypes, unclassifiable (UNC) DLBCL, primary mediastinal B cell lymphoma (PMBCL), Burkitt lymphoma (BL), and mantle cell lymphoma (MCL). Using our previous GEP data, diagnostic signatures, nCounter system (Nanostring, Seattle, WA), and employing published procedures (Scott et al, Blood 2014); we designed probes to 800 genes with utility in distinguishing between these pathological entities. The training cohort comprised 107 unique cases, whose FFPET biopsies were independently assayed at the Molecular Characterization Laboratory, Frederick National Laboratory for Cancer Research (Frederick, MD) and the Centre for Lymphoid Cancer, BC Cancer Agency (Vancouver, BC). The resulting algorithm was locked down and applied to an independent cohort of 199 cases. The nucleic acids from FFPET biopsies from these cases were extracted and run across the two independent laboratories with 83 cases run at both laboratories to assess inter-laboratory performance. The gold standard by which the Nanostring classification was compared was based on Affymetrix gene expression profiling of matched frozen biopsies in the cases of ABC, GCB, and UNC DLBCL (Lenz et al. NEJM 2008) and on the pathological diagnosis by the Hematopathology review panel in the cases of BL, MCL, and PMBCL. The use of human tissues and clinical data for this study was approved by the University of Arizona Institutional Review Board in accordance with the Declaration of Helsinki. Results: The final locked algorithm consisted of 297 gene probes including 47 housekeeping genes. Thirty-six cases from the training cohort were run again on the new lot of Nanostring code set to allow for cross code set calibration of the assay. The laboratory procedure and algorithm, together termed the "Lymph5Cx" test, consists of a hierarchical series of pair-wise comparisons. In the independent validation set, 257/282 assays (91.1%) yielded gene expression data of sufficient quality (total of 185 of the 199 cases). A classification summary is given in the Table below. In this cohort, 136 cases (82%) were correctly assigned while 11 cases (6.6%) were assigned incorrect diagnoses as follows: 6 BL assigned to GCB, 1 GCB labeled a PMBCL, and 4 PMBCL assigned to DLBCL subtypes. The Lymph5Cx test included categories of indeterminate results between two diagnostic entities and were declared borderline, as indicated in the Table. The agreement between the 2 laboratory sites was 71/72 (99%) of cases that yielded adequate gene expression data at both sites. Conclusions: The Lymph5Cx test was robust and able to discriminate the often clinically difficult diagnostic categories of agg-B-NHL using a single methodology for cases with histologic and immunophenotypic features of an agg-B-NHL. Misclassification errors were low, suggesting that this test would be useful adjunct to current diagnostic methods. In addition, targetable pathways, as well as genes associated with known prognostic signatures in DLBCL (stromal) and MCL (proliferation) were quantified. Investigation into these latter aspects is on-going. Gene expression signature assays have become a useful clinical and research tool in the on-going area of precision therapeutics based on highly-defined molecular entities. Table # cases % accurate % borderline % error ABC 26 76.9% 23.1% 0.0% GCB 27 88.9% 7.4% 3.7% BL 48 68.8% 19.8% 11.5% PMBL 30 80.0% 6.7% 13.3% MCL 34 100.0% 0.0% 0.0% Disclosures Scott: Nanostring: The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Wright:Nanostring: The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Williams:Nanostring: The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Lih:Nanostring: The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Jaffe:Nanostring: The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Rosenwald:Nanostring: Research Funding, The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Campo:Nanostring: Research Funding, The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Chan:Nanostring: The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Connors:Nanostring: The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Smeland:Nanostring: The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Braziel:Nanostring: Research Funding, The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Ott:Nanostring: The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Delabie:Nanostring: Research Funding, The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Weisenburger:Nanostring: The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Cook:Nanostring: Research Funding, The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Greiner:Nanostring: The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Fu:Nanostring: Research Funding, The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Walsh:Nanostring: The author is a potential inventor on a patent application using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Gascoyne:Nanostring: Research Funding, The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Staudt:Nanostring: The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Rimsza:Nanostring: Research Funding, The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties.

Description: Abstract 686 Background DLBCL has two molecular subtypes, termed activated B cell-like (ABC) and germinal center B cell-like (GCB), with ABC DLBCL being less curable with current therapy. The survival of ABC but not GCB DLBCL cell lines is sustained by “chronic active” B cell receptor (BCR) signaling. Gain-of-function mutations affecting the BCR subunit CD79B occur in 21% of ABC but only 5% of GCB DLBCL tumors. ABC DLBCL cell lines also depend upon a second pathway for survival that is coordinated by MYD88, an adapter for Toll-like receptors. A constitutively active MYD88 mutant (L265P) is frequent in ABC DLBCL tumors (29%) but rare in GCB DLBCL. CD79B and MYD88 L265P mutations often coexist in ABC DLBCL tumors, suggesting oncogenic collaboration, but can also occur alone. Here, we present interim results of a phase 2 study in relapsed/refractory DLBCL of ibrutinib, a first in class inhibitor of BTK, a kinase in the BCR pathway. We tested the hypothesis that ibrutinib would be more active in ABC than GCB DLBCL due to their different addiction pathways, and we assessed the association of CD79B and MYD88 mutations with response. Methods Subjects with relapsed/refractory de novo DLBCL received ibrutinib 560 mg PO QD. Gene expression profiling (GEP) of formalin-fixed paraffin-embedded biopsy tissues using Affymetrix arrays was used to identify the DLBCL subtype (ABC, GCB, unclassifiable) or were not arrayed (unknown). Sanger sequencing was used to identify CD79B and MYD88 mutations. Subjects underwent CT and PET scanning pre-treatment and every 2 cycles. The primary objective of the study was overall response rate (ORR) categorized by molecular subtype. Response was investigator determined using the revised International Working Group Criteria for NHL. Subjects Seventy subjects were enrolled; median age 63 yrs (28–92); male 71%; stage IV 63%; HI-I/HI IPI 59%; disease ≥10 cm 23%; median prior systemic therapies 3 (1–7); relapsed (27%), refractory (54%) and unknown (19%); prior stem cell transplant 23%; and median time from diagnosis 19 months. Results Safety data are available for 68 subjects who received ≥ 1 dose of ibrutinib. Ibrutinib was well tolerated, with treatment-emergent AEs consistent with data reported in other ibrutinib studies. No new safety signals were identified. Sixty subjects were evaluable per protocol for response (≥ 1 dose of ibrutinib and at least one response assessment). Four subjects in the ABC cohort were not evaluable for response (1 death at study day 12, 1 PD at study day 65, and 2 remain on study treatment but have not had their first response assessment at this analysis). One subject in the GCB cohort was not evaluable for response (death at study day 41). In the ABC subtype, per protocol ORR was 40% (10/25, 95% CI: 21–61%), CR 8% (2/25) and PR 32% (8/25). The median PFS at the time of this analysis is 5.5 months in ABC responders with 60% having not progressed (5 remain on treatment and 1 responder proceeded to transplant). Only one PR was observed in the GCB subtype and none in unclassifiable cases. Thus, ibrutinib showed preferential response activity in ABC versus GCB DLBCL (p=0.0126, Fisher's exact test). Responses occurred in ABC DLBCL tumors with CD79B mutations (60%; 3/5), but also in those with wild type CD79B (37%; 7/19), suggesting that ibrutinib sensitivity does not require a BCR mutation. All cases with both CD79B and MYD88 L265P mutations (n=4) responded, showing that the MYD88 pathway does not prevent ibrutinib activity. In comparison, tumors with only a MYD88 L265P mutation (n=4) did not respond (p=0.0286, Fisher's exact test), suggesting a MYD88-dependent but BCR-independent pathogenesis for some ABC DLBCL cases. Conclusions Ibrutinib showed a clinically meaningful response rate in relapsed/refractory ABC DLBCL, but not in other molecular subtypes. These results are consistent with an essential role of BCR signaling in ABC DLBCL and indicate that future clinical trials of ibrutinib in DLBCL should enroll patients with this subtype. Disclosures: Goy: Pharmacyclics: Research Funding. de Vos:Pharmacyclics: Research Funding. Kenkre:Pharmacyclics: Research Funding. Blum:Pharmacyclics: Research Funding. Advani:Phramacyclics, Inc: Research Funding. Kunkel:Pharmacyclics, Inc: Employment, Equity Ownership. McGreivy:Pharmacyclics, Inc.: Employment, Equity Ownership. Balasubramanian:Pharmacyclics, Inc.: Employment, Equity Ownership. Cheng:Pharmacyclics, Inc.: Employment, Equity Ownership. Moussa:Pharmacyclics, Inc.: Employment, Equity Ownership. Buggy:Pharmacyclics, Inc.: Employment, Equity Ownership.

Description: The diffuse large B-cell lymphoma (DLBCL) cell-of-origin (COO) distinction into germinal center B cell (GCB) and activated B cell (ABC) subtypes, as molecularly described by our group, has profound biological, prognostic, and potential therapeutic implications. New therapeutic agents with selective activity in ABC and GCB DLBCL are under development. An accurate diagnostic assay is urgently needed to qualify patients for clinical trials using targeted agents and as a predictive biomarker. Although the subtypes were originally defined using gene expression profiling on snap-frozen tissues (frozen-GEP), it has become common practice to use less precise but relatively inexpensive and broadly applicable immunohistochemical (IHC) methods in formalin-fixed paraffin-embedded tissue (FFPET). We sought to create a robust, highly accurate molecular assay for COO distinction using new GEP techniques applicable to FFPET. Studies were performed on centrally reviewed DLBCL FFPET biopsies using cases that had “gold standard” COO assigned by frozen-GEP using Affymetrix U133 plus 2.0 microarrays. The training cohort consisted of 51 cases comprising 20 GCB, 19 ABC and 12 Unclassifiable (U) cases. An independent validation cohort, consisting of 68 cases (28 GCB, 30 ABC, 10 U) drawn from the validation cohort of Lenz et al (NEJM 2008) had the typical proportions of COO subtypes seen in DLBCL populations. Nucleic acids were extracted from 10um FFPET scrolls. Digital gene expression was performed on 200ng of RNA using NanoString technology (Seattle, WA). All FFPET GEP studies were performed in parallel at two independent sites (BC Cancer Agency, Vancouver, and NCI, Frederick, MD) using different FFPET scrolls to determine inter-site concordance and assess the robustness and portability of the assay. To assign COO by IHC, tissue microarrays were made using 0.6mm duplicate cores from 60/68 validation cohort cases, and stained for CD10, BCL6, MUM1, FOXP1, GCET1 and LMO2. Two hematopathologists independently assessed the proportion of tumor cells stained, with consensus on discordant cases reached with a third hematopathologist. For the validation studies, those producing and analyzing the GEP and IHC data were blinded to the “gold standard” COO. All 119 FFPET biopsies yielded sufficient RNA. A pilot study using the training cohort identified 20 genes (15 genes of interest and 5 house keeping genes) whose expression, measured using NanoString, would allow accurate replication of the COO assignment model of Lenz et al (NEJM 2008). NanoString was then used to quantitate these 20 genes in the training cohort, allowing the COO model to be optimized. Despite the age of the FFPET blocks (6-32 years old), 95% (49/51) of the training samples gave gene expression data of sufficient quality. The model, including coefficients, thresholds and QC parameters was then “locked” and applied to the independent validation cohort. Ninety-nine percent (67/68) of the samples from the validation cohort (5-12 years old) provided gene expression of adequate quality. Three cases did not give interpretable IHC results. When considering the “gold standard” ABC and GCB cases, the COO assignments by the NanoString assay at the NCI site were 93% concordant, with 5% labeled U and 1 ABC misclassified as GCB (see table). This 2% rate of misclassification of ABC and GCB cases compares favorably with the 9%, 6% and 17% rates for the interpretable cases from the Hans, Tally and Choi algorithms, respectively. Furthermore, the 98% concordance of COO assignment (95% if “gold standard” U cases are also included) between the NCI and BC Cancer Agency sites indicates that, in contrast to the IHC algorithms, the assay is reproducible.TableNanoString GEP assay - NCIHans algorithmTally algorithmChoi algorithmGCBUABCGCBNon-GCBGCBABCGCBABCFrozen GEPGCB2800210183192U721552864ABC1325422026620 In summary, 119 well-characterized DLBCL cases from the LLMPP, previously subtyped by our published disease-defining algorithm using frozen-GEP, were used to develop a highly accurate and robust NanoString 20 gene assay, applicable to RNA from FFPET that is routinely obtained for diagnosis. This new assay shows excellent performance in archival FFPET, and the rapid turn-around time (