Citations

Abstract Introduction - With over 12,000 deaths from MM anticipated in 2016, nearly all patients (pts) with multiple myeloma (MM) will become "quad refractory" to IMIDs (lenalidomide and pomalidomide) and proteasome inhibitors (bortezomib and carfilzomib), and eventually "penta refractory" to anti-CD38 Abs (daratumumab and isatuximab), defining high unmet need populations. Selinexor, an oral selective XPO1 inhibitor, induces nuclear accumulation and activation of tumor suppressor proteins, inhibition of NF-kB, and inhibition of translation of several oncoprotein mRNAs such as c-myc and cyclin D. Selinexor showed potent induction of apoptosis of MM cells independent of p53 signaling. In phase 1 clinical studies, selinexor with low dose dexamethasone (Sd) demonstrated potent anti MM activity in pts with MM. Methods - This phase II clinical trial evaluated Sd in pts with MM refractory to bortezomib, carfilzomib, lenalidomide, and pomalidomide ("quad"), with a subset also refractory to an anti-CD38 Ab ("penta"). Inclusion required CrCL≥20 mL/min, ANC≥1000/µL, platelets ≥50K/µL (≥30K if plasma cells were ≥50% of marrow cellularity). Pts were treated twice weekly (BIW) with oral selinexor 80 mg for 6 or 8 doses per 28 day cycle and dexamethasone (dex) 20 mg BIW. All pts received 5-HT3 antagonists. The primary objective was to determine the overall response rate (ORR) per IMWG criteria and duration of response (DOR), both adjudicated by an independent review committee (IRC). Secondary endpoints include progression free survival (PFS) and overall survival (OS). FISH analyses and gene expression profiling were performed on bone marrow aspirates. Results - 79 pts were enrolled: 48 quad (24 M/24 F, median age 62 yrs) and 31 penta (13 M / 18 F, median age 68 yrs). Both groups had a median of 7 prior treatment regimens including multiple dex-containing regimens. Baseline laboratory abnormalities included grade (Gr)≥3 anemia in 13% and Gr≥3 thrombocytopenia in 8%. Most penta pts received 8 doses / cycle (65%); most quad pts received 6 doses / cycle (83%). Common treatment-related adverse events (TRAEs) hematological: thrombocytopenia (72%, Gr 3/4 58%), anemia (48%, Gr 3 25%) and neutropenia (29%, Gr≥3 21%). TRAEs non-hematological: nausea (72%, Gr 3 6%), fatigue (62%, Gr 3 14%) anorexia (49%, Gr 3 3%), vomiting (43%, Gr3 4%), asymptomatic hyponatremia (42%. Gr 3 20%), diarrhea (42%, Gr 3 5%) and weight loss (33%, Gr 3 1%). There was one case of febrile neutropenia (1%) and one case of clinically significant bleeding related to thrombocytopenia (1%). Seventy pts have discontinued therapy: PD (73%), AEs (17%), physician/pt preference (1%) and 6 deaths (one case related to selinexor, intracranial bleed in pt with Gr4 thrombocytopenia). Nine pts remain on study. Efficacy was evaluated in 78 pts (1 pt did not have measurable disease). The IRC-determined ORR (≥PR) for all pts was 21%, including 5% VGPR. ORR was 21% for quad pts and 20% for penta pts. Clinical benefit rates (≥MR) were 32% (all), 29% (quad), and 37% (penta). Median OS was 9.3 months (mo) for all pts, >11 mo (median not reached) for responders (≥PR), and 5.7 mo for non-responders. Median DOR in responding pts was 5 mo, and median PFS in all pts was 2.1 mo. Baseline cytogenetics were assessed in 41 pts. The ORR in 18 pts with high-risk FISH abnormalities was 33% (Table 1). Notably, 3 of the 13 pts with a 17p abnormality responded (ORR 23%). Transcriptomic profiling revealed differentially expressed genes (DEGs) between responders and non-responders in both whole blood RNA and CD138+ bone marrow cells. Pathways enriched in responders included IL-6, IL-8 and IGF-1 pathways. Conclusions - Oral Sd is active in heavily pretreated pts with refractory MM, including those with MM refractory to anti-CD38 Ab and those with high-risk cytogenetic abnormalities. Response was associated with longer survival. The main toxicities of Sd are thrombocytopenia, nausea, anorexia, and fatigue. AEs were manageable with supportive care and dose interruptions/reductions. To our knowledge, this is the first report of anti-tumor activity in the penta-refractory MM population. This population of MM pts has exhausted all currently available treatment options and has an extremely poor prognosis and therefore requires new therapies. Expansion of this trial in this high unmet medical need, penta refractory population is planned. Table 1 Activity of Sd in Patients with High Risk MM Cytogenetics Table 1. Activity of Sd in Patients with High Risk MM Cytogenetics Disclosures Vogl: Constellation: Research Funding; Karyopharm: Consultancy; Acetylon: Research Funding; GSK: Research Funding; Calithera: Research Funding; Teva: Consultancy; Celgene: Consultancy; Takeda: Consultancy, Research Funding. Jagannath:Bristol-Myers Squibb: Consultancy; Janssen: Consultancy; Novartis: Consultancy; Merck: Consultancy; Celgene: Consultancy. Baz:Bristol-Myers Squibb: Research Funding; Takeda/Millennium: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Signal Genetics: Research Funding; Novartis: Research Funding; Karyopharm: Research Funding; Merck: Research Funding. Nooka:Spectrum, Novartis, Onyx pharmaceuticals: Consultancy. Richter:Takeda: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Janssen: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Novartis: Speakers Bureau. Vij:Karyopharm: Honoraria; Amgen: Honoraria, Research Funding; Celgene: Consultancy; Takeda: Honoraria, Research Funding; Novartis: Honoraria; Bristol-Myers Squibb: Honoraria; Janssen: Honoraria. Schiller:Incyte Corporation: Research Funding. Costa:Sanofi: Honoraria, Research Funding. Chari:Array Biopharma: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Pharmacyclics: Research Funding; Janssen: Consultancy, Research Funding; Amgen Inc.: Honoraria, Research Funding; Novartis: Consultancy, Research Funding. Siegel:Takeda: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Celgene: Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau; Merck: Honoraria. Fonseca:Janssen: Consultancy; AMGEN: Consultancy; Millennium, a Takeda Company: Consultancy; Bayer: Consultancy; Sanofi: Consultancy; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; AMGEN: Consultancy; Novartis: Consultancy; Millennium, a Takeda Company: Consultancy; Sanofi: Consultancy; AMGEN: Consultancy; Janssen: Consultancy; BMS: Consultancy; Celgene: Consultancy; BMS: Consultancy; Millennium, a Takeda Company: Consultancy; Millennium, a Takeda Company: Consultancy; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Bayer: Consultancy; Novartis: Consultancy; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms; AMGEN: Consultancy; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Patent: Patents & Royalties: Prognostication of MM based on genetic categorization of FISH of the disease; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms; Patent Pending: Patents & Royalties: The use of calcium isotopes as biomarkers for bone metabolisms; Celgene: Consultancy. Kauffman:Karyopharm Therapeutics Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Shacham:Karyopharm Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Saint-Martin:Karyopharm: Employment. Picklesimer:Karyopharm: Employment. Friedlander:Karyopharm Therapeutics: Employment. Choe-Juliak:Karyopharm Therapeutics: Employment.

Abstract Abstract 788 FLT3 mutations are common in AML and are associated with poor prognosis. Lestaurtinib is a multi-targeted kinase inhibitor with potent activity against FLT3. In an attempt to definitively establish whether or not there is clinical benefit for FLT3 inhibition in the relapse setting, we conducted a randomized trial of chemotherapy followed by lestaurtinib for patients with FLT3 mutant AML in first relapse. Patients were randomized to receive either chemotherapy alone, consisting of mitoxantrone, etoposide, and cytarabine (MEC) or high-dose cytarabine (HiDAc) depending on the duration of CR1) or chemotherapy followed by 80 mg lestaurtinib orally BID. Patients on the chemotherapy only arm were eligible for crossover if they were refractory to therapy. Patients on the lestaurtinib arm received the drug for up to 112 days on study (they could continue to receive the drug through a separate extension protocol). The primary objective was complete remission (CR/CRp). Secondary objectives included overall survival (OS), safety, and tolerability. From January 2004 through December 2008, 224 patients at 52 different centers were enrolled and randomized, with 220 patients actually receiving therapy. Blood samples from patients on the lestaurtinib arm were obtained at baseline, on Day 15, and on Day 42 for pK studies and for determination of in vivo FLT3 inhibition and measurement of plasma FLT3 ligand (FL) and alpha-1 acid glycoprotein (AGP) levels. The median age for all patients enrolled was 55 years (53.5 in the control arm, 58.5 in the lestaurtinib arm), with 88% having FLT3/ITD mutations, 8% having D835 mutations, and 4% having both. The duration of CR1 was less than 6 months in 47% of patients in each arm. On the lestaurtinib arm, 107 patients received at least 1 week of lestaurtinib (mean 60.1 days, range 8-418), with 17 of these patients receiving drug on the extension protocol. On the chemotherapy only arm, 7 patients crossed over to receive lestaurtinib, and 31 patients went on to receive lestaurtinib on the extension protocol after outcome evaluation. Lestaurtinib was generally well tolerated following chemotherapy, with 104 Grade 3/4 adverse events in the lestaurtinib arm versus 99 in the control arm. There were 61 serious adverse events in the lestaurtinib arm versus 49 in the control arm. Early death (by Day 42) occurred in 22 lestaurtinib patients (2 progressive leukemia, 14 infectious, 6 organ failure) and16 control patients (6 progressive leukemia, 5 infectious, 5 organ failure). By intention to treat analysis, there were 29 CR/CRp in the lestaurtinib arm and 23 in the control arm (26% versus 21%; p = 0.35), with no difference in OS (4.73 months versus 4.57 months) between the two arms. Blood samples from 79 of the 107 lestaurtinib-treated patients obtained on Day 15 were analyzed for FLT3 inhibitory activity (plasma inhibitory activity [PIA] assay), as well as FLT3 ligand (FL), lestaurtinib, and AGP concentrations. Mean lestaurtinib plasma concentration was 12 uM at Day 15 and fell to 7.5 uM on Day 42. Mean lestaurtinib concentration at Day 15 was 13.3 uM in patients achieving CR/CRp and 11.5uM in non-responders. A target of >85% inhibition of FLT3 maintained at trough (pre-dose) was defined from previous studies. Of the 79 patients tested, 46 (58%) achieved this degree of FLT3 inhibition on Day 15. FL concentrations rose from baseline (15.6 pg/mL) to Day 15 (1148 pg/mL), and AGP concentrations rose by an average of 52% over the same period, both of which may have reduced the degree of FLT3 inhibition. Of the 46 patients with target FLT3 inhibition on Day 15, 18 (39%) achieved CR/CRp, while only 3 (9%) of the 32 patients with below target FLT3 inhibition achieved CR/CRp. Thus, in patients with FLT3 mutant AML at first relapse, pharmacokinetic factors and possible physiologic factors (FL and AGP) limit lestaurtinib's ability to effectively inhibit FLT3. FLT3 inhibition by lestaurtinib, when achieved, correlates with better CR rates, but in this trial that benefit was negated by a poor CR rate in those patients for whom the drug did not reach the target level of FLT3 inhibition. Overall, lestaurtinib treatment following re-induction chemotherapy failed to increase response rates or prolong survival of patients with FLT3 mutant AML in first relapse. Disclosures: Levis: Cephalon: Clinical Advisory Board member. Ravandi:Cephalon: Honoraria, Member, clinical advisory board, Research Funding. Erba:Cephalon: Research Funding, Speakers Bureau. Baccarani:Novartis Pharma, Bristol Myers Squibb, Merck Sharp & Dome, Pfizer: Consultancy, Speakers Bureau. Stone:Cephalon: ad hoc consultancy; Novartis: Research Funding, ad hoc consultancy. Advani:Cephalon: Research Funding. Douer:Cephalon: Honoraria, Research Funding, Speakers Bureau. Litzow:Cephalon: Research Funding. Tremmel:Cephalon: Employment, Equity Ownership. Bensen-Kennedy:cephalon: Employment. Smith:Cephalon: Research Funding.

Abstract Abstract 414 Background: SGI-110 is a dinucleotide of decitabine (DAC) and deoxyguanosine formulated as a low volume and pharmaceutically stable SQ injection allowing more extended decitabine exposure than DAC IV infusion. The anticipated differentiated PK profile offers the potential of improved biological and clinical activity and safety over currently available HMAs. Methods: A randomized Phase 1–2 FIH Pharmacokinetics/Pharmacodynamics (PK/PD)-guided, dose-escalation study has been conducted in patients with relapsed/refractory intermediate or high-risk MDS or AML. The objective of the first stage (dose escalation) is to determine safety and tolerability and to establish the Maximum Tolerated Dose (MTD) and Biologically Effective Dose (BED). Patients were randomized to one of two SQ regimens (dailyx5 or once weeklyx3, 28-day courses). PD is evaluated by LINE-1 global DNA hypomethylation. The second stage (dose expansion) is to determine the clinical activity and safety in AML and MDS using the established BED and MTD. We report here the results of the dose escalation phase which has completed enrolment. Results: 78 patients (64 AML, 14 MDS) were enrolled in the dose escalation phase: 44 patients in the dailyx5 regimen and 34 in the weeklyx3 regimen. Median age was 69 years (range 29–86), 65% were male, and 82% had ECOG PS of 0–1. Median number of prior regimens was 3 (range 1–9), 59% of patients had prior HMA treatment (50% of AML patients, and 100% of MDS patients). Six patients are still ongoing treatment. The PK profile demonstrated efficient conversion of SGI-110 to decitabine as predicted from the SGI-110 rational design, resulting in longer decitabine exposure window (beyond 8 hrs) compared to DAC IV (3–4 hrs). At SGI-110 dose range of 60–125 mg/m2, observed mean decitabine AUCs (88–231 ng*hr/mL) reach or exceed the therapeutic range seen with 20 mg/m2 DAC IV (115 ng*hr/mL) while achieving only a small fraction of the Cmax (26–64 ng/mL vs 146 ng/mL for DAC IV). The effective half-life for decitabine after SQ SGI-110 injection appeared to be prolonged (up to 4-fold or ∼2.4 hrs) compared to DAC IV (0.58 hrs). Decitabine exposures (AUC) increased in a dose-proportional manner regardless of the regimen and no accumulation was observed. Dose-related LINE-1 hypomethylation was observed in patients treated with the daily regimen between 18 and 60 mg/m2; a plateau in maximum average hypomethylation (∼25%) was evident at higher daily doses (90-125 mg/m2) and therefore the BED for the dailyx5 schedule is established at 60 mg/m2. The 25% average hypomethylation of LINE-1 compares favorably with that observed historically after DAC IV at the dose of 20 mg/m2 dailyx5. The extent of LINE-1 hypomethylation after weeklyx3 SGI-110 was inferior as the maximum average hypomethylation plateaued at ∼8% from baseline. Starting at 36 mg/m2 daily and 60 mg/m2 weekly (44 AML, and 7 MDS patients), clinical responses were observed: 2CRs, 1CRp, and 1CRi in heavily pretreated AML patients; 1 mCR and 1 HI in MDS patients previously treated with azacitidine. All responses were in patients who achieved >10% LINE-1 hypomethylation. The most common Adverse Events (AEs), regardless of relationship to SGI-110, were diarrhea (21%), febrile neutropenia (17%), fatigue/injection site pain/nausea at 15% each. The most common drug-related AEs were injection site pain (15%), fatigue (8%), nausea (6%), and thrombocytopenia (5%). MTD was not reached with the weekly regimen up to 125 mg/m2 weeklyx3. With the daily regimen, 125 mg/m2dailyx5 resulted in 2 Dose-Limiting Toxicities (DLTs) of febrile neutropenia in 3 MDS patients (1 associated with bacteremia, and the other with sepsis and thrombocytopenia Grade 4) while none of the 9 patients with AML had DLT at that dose. Conclusions: SGI-110 is well tolerated at doses higher than BED which is established at 60 mg/m2 dailyx5 where average hypomethylation of ∼25% was achieved. MTD estimated to be 90 mg/m2 dailyx5 for MDS and 125 mg/m2dailyx5 for AML patients. SQ administration of SGI-110 achieved efficient conversion to decitabine resulting in an improved PK profile over DAC IV. Clinical responses were observed in this heavily pretreated population and they seem to correlate with the extent of LINE-1 hypomethylation. Study is currently enrolling patients in the dose-expansion Phase 2 stage. Disclosures: Kantarjian: Astex Pharmaceuticals: Research Funding. Roboz:Astex Pharmaceuticals: Research Funding. Rizzieri:Astex Pharmaceuticals: Research Funding. Stock:Astex Pharmaceuticals: Research Funding. O'Connell:Astex Pharmaceuticals: Research Funding. Griffiths:Astex Pharmaceuticals: Research Funding. Yee:Astex Pharmaceuticals: Research Funding. Tibes:Astex Pharmaceuticals: Research Funding. Garcia-Manero:Astex Pharmaceuticals: Research Funding. Ravandi:Astex Pharmaceuticals: Research Funding. Walsh:Astex Pharmaceuticals: Research Funding. Feldman:Astex Pharmaceuticals: Research Funding. Ritchie:Astex Pharmaceuticals: Research Funding. Rao:Astex Pharmaceuticals: Research Funding. Decastro:Astex Pharmaceuticals: Research Funding. Schimmer:Astex Pharmaceuticals: Research Funding. Mesa:Astex Pharmaceuticals: Research Funding. Syed:Astex Pharmaceuticals: Research Funding. Choy:Astex Pharmaceuticals: Employment. Oganesian:Astex Pharmaceuticals: Employment. Taverna:Astex Pharmaceuticals: Employment. Azab:Astex Pharmaceuticals: Employment. Chung:Astex Pharmaceuticals: Research Funding. Issa:Astex Pharmaceuticals: Honoraria, Research Funding.

Susceptibility testing of the polymyxins (colistin and polymyxin B) is challenging for clinical laboratories. The Clinical and Laboratory Standards Institute (CLSI) Antimicrobial Susceptibility Testing Subcommittee evaluated two methods to enable accurate testing of these agents. These methods were a colistin broth disk elution (CBDE) and a colistin agar test (CAT), the latter of which was evaluated using two inoculum volumes, 1 μl (CAT-1) and 10 μl (CAT-10).

Abstract Background: I-SPY2 is a multicenter, Phase II trial using response-adaptive randomization within biomarker subtypes to evaluate novel agents as neoadjuvant therapy for high-risk at least T2N0 breast cancer. The primary endpoint is pathologic complete response (pCR) at surgery. The goal is to identify regimens that have ≥ 85% Bayesian predictive probability of success in a 300-patient phase 3 neoadjuvant trial defined by hormone-receptor (HR) and HER2 status, and MammaPrint (MP). Regimens may leave the trial for futility (< 10% probability of success), maximum sample size accrual (with probability of success ≥ 10% and < 85%), or as recommended by the independent DSMB. For HER2- subjects the control arm is weekly paclitaxel x12 then doxorubicin & cyclophosphamide (AC) q2-3 weeks x4. For this arm, paclitaxel was omitted and replaced with maximum tolerated dose PARPi talazoparib with synergy dosed irinotecan (25mg/m2). Paclitaxel could be given adjuvantly for these subjects and non-responding subjects could be taken off of experimental therapy. Methods: Women with tumors ≥ 2.5cm were eligible for screening. MP low/HR+ tumors were ineligible. MRI scans (baseline, 3 cycles after start of therapy, prior to AC, and prior to surgery) were used in a longitudinal statistical model to predict pCR for individual patients. Talazoparib was given at 1mg daily with 25mg/m2 irinotecan q2wks. Analysis was intention to treat. Subjects who switched to non-protocol therapy count as non-pCR. Subjects on experimental therapy at time of arm closure are non-evaluable. Talazoparib/irinotecan (TI) was open only to HER2- tumors and eligible for graduation in 3 of 10 pre-defined signatures: HER2-, HR+HER2- and HR-/HER2-. Results: TI did not meet criteria for graduation and was stopped at the recommendation of the DSMB based on expectations of limited activity beyond that seen with standard treatment. Maximum sample size had been reached at the time of this recommendation and subjects currently receiving TI were allowed to continue or change to standard therapy. Exploratory “as treated” analysis for response in gBRCA mutation carriers showed 6/10 gBRCA carriers attained a pCR in the TI arm. Except for 1 patient these gBRCA pCR subjects had >90% tumor reduction by MRI after TI and prior to AC (range: 68-96%). In the TI arm pCR rates were also higher in subjects with a PARPi7-High/MP2 gene expression signature (0.344 vs 0.146). Expected differences in toxicity were seen between arms including g3/4 peripheral neuropathy on control therapy which included paclitaxel (2.6% vs none) and g3/4 neutropenia with TI (30.2% vs 8.2%). Notably gBRCA mutation carriers receiving TI had higher rates of g3/4 neutropenia (60% vs 25.9%). Conclusion: The I-SPY2 study finds the probability that investigational regimens will be successful in a Phase III neoadjuvant trial; TI did not reach the efficacy threshold of 85% probability of success in Phase III in any of the 3 signatures. However by adding talazoparib with synergy dosed irinotecan we were able to omit paclitaxel and observe similar estimated pCR rates. This informs current work to evolve the I-SPY2 trial design to reduce toxicity without compromising outcomes and develop successful combinations targeted to biology, including DNA repair deficiency. Citation Format: Richard Schwab, Amy S. Clark, Christina Yau, Nola Hylton, Wen Li, Denise Wolfe, A Jo Chien, Anne M. Wallace, Andres Forero-Torres, Erica Stringer-Reasor, Rita Nanda, Nora Jaskowiak, Judy Boughey, Tufia Haddad, Heather S. Han, Catherine Lee, Kathy Albain, Claudine Isaacs, Anthony D. Elias, Erin D. Ellis, Payal Shah, Julie E. Lang, Janice Lu, Debasish Tripathy, Kathleen Kemmer, Douglas Yee, Barbara Haley, Melanie Majure, Erin Roesch, Christos Vaklavas, Cheryl Ewing, Teresa Helsten, W Fraser Symmans, Jane Perlmutter, Hope S. Rugo, Michelle Melisko, Amy Wilson, Ruby Singhrao, Laura van 't Veer, Angela DeMichele, Smita Asare, Don Berry, Laura J. Esserman. Evaluation of talazoparib in combination with irinotecan in early stage, high-risk HER2 negative breast cancer: Results from the I-SPY 2 TRIAL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT136.

Abstract Background: Pembrolizumab (Pembro), an anti-PD-1 immune checkpoint inhibitor, has been approved for the treatment of a variety of cancers. Pembro was recently evaluated in HER2- breast cancer patients in the neoadjuvant I-SPY 2 TRIAL and graduated in the triple negative (TN), HR+HER2-, and HER2- signatures. TN breast cancers tend to have high numbers of immune infiltrates, including T cells and tumor associated macrophages (TAMs). We utilized multiplex multispectral imaging to evaluate the presence and spatial proximity of various immune cell populations as specific predictors of response to Pembro. Methods: Pre-treatment biopsies were available from 54 patients. FFPE sections were immunostained using Opal fluorescence reagent kits on a fully automated Ventana Discovery platform and imaged with a Vectra automated imaging system. Image analyses were performed with a variety of software packages. Immune biomarkers included CD3, CD8, FoxP3, CD68, PD-1, PD-L1, and cytokeratins. Cell densities were determined per area of tissue. Spatial analyses were performed to quantitate proximity of cell types. We used logistic regression with the likelihood ratio test to evaluate associations between pathologic complete response (pCR) with immune cell counts, ratios, and spatial proximity measurements. This analysis was also performed adjusting for HR status, and within receptor subsets, sample size permitting. Our statistics are descriptive rather than inferential and do not adjust for multiple hypothesis testing or multiplicities of other biomarkers outside this study. Results: Densities of CD3+ T cells ranged from 193-5155 cells/mm2, with CD8+ cytotoxic T cells (Tc) ranging from 103 to 3774 cells/mm2 and FoxP3+ Tregs from 0-416 cells/mm2. Densities of PD-1+ T cells and PD-1+ Tc cells ranged from 0-1341 cells/mm2 and 0-698 cells/mm2, respectively. Macrophage cell densities ranged from 84-2644 cells/mm2. PD-L1+ tumor cell densities ranged from 0-2981 cells/mm2. High PD-L1 expression (>=10% of tumor cells positive for PD-L1) was observed in 12% of the cases. Overall T cell density was positively associated with pathological complete response (pCR), as were Treg cell density and PD-1+ T cell density. The ratio of macrophages to Tc cells was negatively associated with response. Finally, the spatial distribution of CD3+ T cells in proximity to cancer cells correlated positively with pCR. Conclusion: Cell density, immune cell ratios and spatial resolution provide insight to response and resistance. We confirmed the observation that T cell density predicts pCR, but more specifically that Treg and PD1+ T cell densities increase the chance of pCR. An increase in macrophages relative to cytotoxic T cells predicts resistance. The closer the T cells are to the tumor, the better the response. These results demonstrate the utility of characterizing the immune microenvironment, in the context of therapy with immuno-oncology agents, not only to predict response, but to gain insight and learn now to potentiate agents and combinations. Citation Format: Michael J. Campbell, Christina Yau, Jennifer Bolen, Scott Vandenberg, Clifford Hoyt, Lamorna Brown-Swigart, Gillian Hirst, Rita Nanda, Minetta Liu, Smita Asare, Laura van'tVeer, Douglas Yee, Angie DeMichele, Don Berry, Laura Esserman, I SPY 2 TRIAL Investigators. Analysis of immune cell infiltrates as predictors of response to the checkpoint inhibitor pembrolizumab in the neoadjuvant I-SPY 2 TRIAL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT003.

Abstract The goal of this study is to evaluate the potential use of gene expression profile of peripheral blood mononuclear cells (PBMCs) as a non-invasive biomarker for predicting the response of anti-PD-1 therapy in non-small cell lung cancer (NSCLC) subjects. Peripheral blood of NSCLC stage 4 subjects treated with an anti-PD-1 monoclonal antibody was collected prior to treatment (week 0) and subsequent infusions. Total RNA was extracted, and libraries were prepared using TruSeq Stranded mRNA LT Sample Prep Kit and sequenced by Illumina NextSeq. The paired-end reads were mapped to human reference hg38 using STAR and gene expressions were calculated using RSEM. Preliminary analysis of differential gene expression (DE) was performed among the responders at respectively (T1) weeks 3-4, (T2) weeks 6-9 and (T3) weeks 12-16 after treatment, compared with the baseline at T0. Further DE analysis was performed between the baseline samples of responders and non-responders. Genes with FDR-adjusted p values less than 0.05 were selected for pathway enrichment analysis with DAVID. Altogether 30 RNA-Seq samples of seven subjects were collected at multiple time points before and after treatment. According to standard RECIST 1.1 criteria, five subjects had a partial response to the therapy, whereas two subjects had stable disease or no response. In addition, liquid biopsy results were available for selected samples. Among the responders, there are 203 DE genes for T1 with log2 fold change (LFC) of gene expression from -2.4 to 3.5, 859 DE genes for T2 with LFC from -3.4 to 3.3, and 395 DE genes for T3 with LFC from -3 to 6.8. Among the DE genes for the three time points, 155 genes are common to T1/T2, 250 common to T2/T3, and 104 common to T1/T2/T3. Pathway enrichment test using Gene Ontology Biological Process terms shows that the strongest DE pathways for initial response after treatment at T1 are regulation of immune response (FDR adjusted p = 2.5E-5), oxygen transport (p = 5.5E-3) and T cell costimulation (p = 3.9E-3). Based on the 104 common DE genes, oxygen transport (p = 0.029) is the only pathway consistently perturbed due to an increase in hemoglobin. When comparing the baseline of responders and non-responders, we identified a candidate gene signature, with some of the genes also reported in other studies. However, more samples are required to establish its statistical significance. Systemic immune response after first dose of anti-PD1 treatment is reflected by DE genes in blood samples collected in weeks 3-4. Tumor regression might be indicated by the consistent increase in hemoglobin level. The results of these analyses show promise of developing a predictive signature for the response of anti-PD-1 immunotherapy in NSCLC subjects based on the gene expression profile of PBMCs. While these are preliminary results, we continue to recruit subjects, and will present the up-to-date results at the conference. Citation Format: Yee Him Cheung, Jie Wu, Weihua Huang, Changhong Yin, Katherine Linder, Kaushal Parikh, Ke Tang, Christopher Kasbek, Jun Huang, Zeil Rosenberg, Michael Fanucchi, John Fallon, Nevenka Dimitrova. Differential gene expression analysis reveals pathways involved in anti-pd-1 therapy response prediction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4994.

Abstract Background: Triple negative breast cancer (TNBC), generally considered aggressive with few options beyond standard chemotherapy, is currently experiencing a period of optimism, with multiple trials showing increased efficacy of platinum containing regimens (with and without PARP-inhibition), and more recently, immunotherapy. In I-SPY 2, veliparib/carboplatin (VC) and the immune checkpoint inhibitor pembrolizumab (Pembro) graduated in the TN subtype. As part of the I-SPY 2 biomarker program, we previously identified the signature PARPi7, a measure of DNA repair deficiency (DRD), as predicting response to VC. As well, immune signatures, including one representing dendritic cells, predicted response to Pembro. Here we investigate the overlap between these biomarkers to identify patient subgroups more likely to respond to immunotherapy or platinum-based therapy and estimate response rates to VC and Pembro in patients positive for one, both, or neither biomarker. Methods: 153 TNBC patients (Control: 85; VC: 39; Pembro: 29) were considered in this analysis. Continuous DNA repair deficiency and immune signatures assayed at the pre-treatment time point were evaluated as published (Wolf, 2017; Danaher, 2017). To identify optimal dichotomizing thresholds, 2-fold cross-validation was repeated 500 times. Bayesian logistic regression was used to estimate pCR rates by arm for patient subsets defined by biomarker combinations. Our study is exploratory with no claims for generalizability; and this analysis does not adjust for multiplicities. Results: Using the optimal dichotomizing cutpoints, 54% of TNBC patients are classified as DRD+, and 67% Immune+. As expected, DRD+ patients have a high estimated pCR rate to VC (75%); and Immune+ patients have a high estimated pCR rate to Pembro (82%). Combining these biomarkers, 40% of TNBC are positive for both biomarkers, 40% for only one biomarker (26% Immune+/DRD-, 14% Immune-/DRD+), and 20% for neither. The Immune+/DRD+ TN subset predicted sensitive to both VC and Pembro has a higher estimated pCR rate to both VC and Pembro relative to control (estimated pCR rates: VC: 75%, Pembro: 73% vs. control: 18%). In contrast, the Immune+/DRD- group, predicted to be sensitive to Pembro, has the highest pCR rate to Pembro (VC: 48%, Pembro: 83% vs. control 21%), whereas the Immune-/DRD+ group is most responsive to VC (VC: 63%, Pembro: 41% vs. control 29%). For the 20% of Immune-/DRD- TNBC patients, their estimated response rates are lower than the biomarker-positive patients (VC: 27%, Pembro: 43% vs. control: 19%). Conclusion: Molecular phenotypes capturing immune activation and DNA repair deficiency may predict response of TNBC and help prioritize selection of platinum or immunotherapy containing regimens. Citation Format: Denise M. Wolf, Christina Yau, Julia Wulfkuhle, Emanuel Petricoin, Lamorna Brown-Swigart, Gillian Hirst, Smita Asare, I-SPY 2 Consortium, Douglas Yee, Angela DeMichele, Hope Rugo, Olufunmilayo Olopade, Rita Nanda, Minetta Liu, Laura Esserman, Laura van t' Veer. Integration of DNA repair deficiency and immune biomarkers to predict which early-stage triple-negative breast cancer patients are likely to respond to platinum-containing regimens vs. immunotherapy: The neoadjuvant I-SPY 2 trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2679.

Abstract Background: In women with breast cancer receiving neoadjuvant chemotherapy, residual cancer burden (RCB) predicts distant recurrence and survival. In those with high risk tumors, locoregional recurrence (LRR) remains a concern, and has been associated with type of local therapy received. We evaluated the impact of local therapy on LRR in the ISPY-2 TRIAL. Methods: Data were analyzed in Stata 14.2, using Chi2 test, log rank test, and a Cox proportional hazards model. RCB was considered a categorical variable (0/1 versus 2/3), as described in prior publications. Breast surgery categories were lumpectomy +/- radiotherapy, or mastectomy +/- radiotherapy. Axillary surgery was defined as sentinel lymph node (SLN) surgery (≤6 nodes removed) or axillary dissection (>6 nodes). Results: Follow up data from the I-SPY2 TRIAL were available for 630 patients (median follow up 2.76 yrs, range 0.4-7.2). Type of local therapy was significantly associated with clinical stage at presentation, with stage III patients most frequently undergoing mastectomy + radiation (p<0.001). Women with higher RCB were more likely to undergo mastectomy than those with lower RCB (61.3% vs 48.8% mastectomy rate, p=0.002), and more likely to receive adjuvant radiotherapy (62.0% vs 53.9%, p=0.048). There was no association between clinical stage, type of surgery, or radiotherapy and LRR (Table). Higher RCB was significantly associated with LRR, with 3 year locoregional recurrence free rate of 95.1% in RCB 0/1 versus 89.9% in RCB 2/3 (p=0.003). In a Cox model adjusting for clinical stage, tumor subtype, surgical therapy, RCB status, nodal radiation, and age, significant predictors for LRR were tumor subtype and RCB status. Hazard ratio (HR) for LRR in those with RCB 0/1 was 0.39 compared to those with RCB 2/3 (95% CI 0.17-0.87, p=0.021). There was no difference in LRR between breast conservation and mastectomy; within the breast conservation group, those who had lumpectomy alone had higher hazard of LRR compared to those having lumpectomy + radiation (HR 3.1, 95% CI 1.1-9.2, p=0.043). Conclusions: Extent of surgical therapy was not associated with local tumor control, regardless of advanced tumor stage at presentation. Rather, tumor biology and response to therapy were the best predictors of LRR. These data highlight the opportunity to minimize the morbidity of extensive surgical therapy for patients with excellent response to systemic therapy. LRR rates by clinical features and treatment status FrequencyLRR RateP valueClinical Stage 0.5I240 (47.5%)5.8% II185 (36.6%)8.7% III80 (15.8%)6.3% Tumor Subtype 0.014ER+PR+Her2-161 (26.4%)3.1% ER+PR-Her2-56 (9.2%)3.6% Her2+176 (28.9%)6.3% Triple negative216 (35.5%)11.1% Local therapy 0.169Lumpectomy85 (13.5%)11.8% Lumpectomy with radiation198 (31.4%)5.6% Mastectomy173 (27.5%)5.2% Mastectomy with radiation174 (27.6%)8.6% Axillary surgery 0.23None5 (0.8%)20% SLN329 (52.2%)5.8% ALND296 (47%)8.5% Axillary radiation 0.535Yes42 (6.7%)9.5% No588 (93.3%)7.0% Axillary management 0.2No surgery or radiation5 (0.8%)20.0% SLN312 (50%)5.3% SLN+Axillary radiation17 (2.7%)8.3% ALND271 (43%)10.3% ALND+Axillary radiation25 (4%)5.4% RCB 0.0020/1293 (50.1%)3.8% 2/3292 (49.9%)10.3% Citation Format: Silverstein J, Suleiman L, Yau C, Price ER, Singhrao R, Yee D, DeMichele A, Isaacs C, Albain KS, Chien AJ, Forero-Torres A, Wallace AM, Pusztai L, Ellis ED, Elias AD, Lang JE, Lu J, Han HS, Clark AS, Korde L, Nanda R, Northfelt DW, Khan QJ, Viscusi RK, Euhus DM, Edmiston KK, Chui SY, Kemmer K, Wood WC, Park JW, Liu MC, Olopade O, Leyland-Jones B, Tripathy D, Moulder SL, Rugo HS, Schwab R, Lo S, Helsten T, Beckwith H, I-SPY 2 TRIAL Consortium, Berry DA, Asare SM, Esserman LJ, Boughey JC, Mukhtar RA. The impact of local therapy on locoregional recurrence in women with high risk breast cancer in the neoadjuvant I-SPY2 TRIAL [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-14-01.

Abstract Background: The explosion in new treatment options targeting immune checkpoints, HER signaling, DNA repair deficiency, AKT, and other pathways calls for updated breast cancer subtypes beyond HR and HER2 status to predict which patients will respond to which treatments. Here we leverage the I-SPY 2 TRIAL biomarker program over the past 8 years across 10 treatment arms to elucidate a minimal set of biomarkers that may improve response prediction in a modern treatment context, and to investigate which new patient phenotypes are identified by these response-predictive biomarkers. Methods: 986 patients were considered in this analysis. Treatments included paclitaxel alone (or with trastuzumab (H) in HER2+) or combined with investigational agents: veliparib/carboplatin (VC); neratinib; MK2206; ganitumab; ganetespib; AMG386; TDM1/pertuzumab (P); H/P; and pembrolizumab (Pembro). 24 prospectively defined, mechanism-of-action and pathway-based expression and phospho-protein signatures/biomarkers assayed from pre-treatment biopsies were previously found to be predictive in a particular agent/arm in pre-specified analysis. Here we evaluate these biomarkers in all patients. We assessed association between each biomarker and response in the population as a whole and within each arm and HR/HER2 subtype using a logistic model. To identify optimal dichotomizing thresholds for select biomarkers, 2-fold cross-validation was repeated 500 times. Our analysis is exploratory and does not adjust for multiplicities. Results: Our initial set of 24 predictive biomarkers reflects DNA repair deficiency (n=2), immune activation (n=7), ER signaling (n=2), HER2 signaling (n=4), proliferation (n=2), phospho-activation of AKT/mTOR (n=2), and ANG/TIE2 (n=1) pathways, among others. Biomarkers reflecting similar biology are correlated and cluster together. We make use of this correlation structure to reduce the dimensionality of the biomarker set to five predictive signals: proliferation, DNA repair deficiency (DRD), immune-engaged (Immune+), luminal/ER (lum), and HER2-activated. These biomarkers, when dichotomized, identify patient groups with differential predicted sensitivities to I-SPY 2 agents and are present at different proportions within receptor subtypes. For instance, in the HER2- subset, Immune+/DRD+ patients are predicted sensitive to both VC and Pembro, and account for 39% of TN, but only 12% of HR+HER2-. On the other end of the spectrum, only 17% of TN are Immune-/DRD-, compared to the majority (56%) of HR+HER2-. There are also subsets of patients positive for only one marker. For the HER2+ subset, 67% are HER2-activated+, and 25% lum+; of these HER2-activated+ patients are more likely to be Immune+ (44%), vs 23% in lum+. HER2-activated+/Immune+ patients have higher predicted sensitivity to HER2-targeted agents than lum+ or Immune- patients. In all, these molecular phenotypes predict sensitivity to one or more I-SPY 2 investigational agents for 75% of the ˜ 1000 patients. Conclusion: Molecular phenotypes reflecting proliferation, immune engagement, HER2-activation, luminal/ER-signaling, and DNA repair deficiency may provide a roadmap to guide treatment prioritization for emerging therapeutics. Citation Format: Wolf DM, Yau C, Wulfkuhle J, Petricoin E, Campbell M, Brown-Swigart L, Hirst G, Asare S, Zhu Z, Lee EP, Delson A, Pohlmann P, I-SPY 2 TRIAL Consortium, Hylton N, Liu MC, Symmans F, DeMichele A, Yee D, Berry D, Esserman L, van 't Veer L. Identifying breast cancer molecular phenotypes to predict response in a modern treatment landscape: Lessons from ˜1000 patients across 10 arms of the I-SPY 2 TRIAL [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-10-02.