Abstract: Introduction Eculizumab (Ecu) is the standard treatment for paroxysmal nocturnal hemoglobinuria (PNH), as it results in sustained control of intravascular hemolysis and prevention of thrombosis, and significant improvement of long-term survival. However, the hematological benefits remain heterogeneous among patients, and most response analyses are restricted to transfusion independence. In the era of new anti-complement therapies, we found appropriate to investigate in detail the quality of hematological response of Ecu-treated PNH patients. Methods We have recently proposed (Risitano et al, Frontiers in Immunology 2019) a classification of hematological response to anti-complement therapies based on hemoglobin (Hb) levels and laboratory markers of hemolysis. Six distinct categories were determined (Table 1): i. complete response (no transfusion requirement with normal stable Hb and no evidence of hemolysis); ii. major response (no transfusion requirement with normal Hb but evidence of residual intravascular or extravascular hemolysis); iii. good response (no transfusion requirement with persistent chronic mild anemia); iv. partial response (persistent chronic moderate anemia and/or with occasional red blood cell transfusions); v. minor response (3-6 red blood cell transfusions every 6 months); vi. no response ( & gt;6 blood cell transfusions every 6 months). Here we have retrospectively exploited this classification to assess hematological response in a large cohort of 93 PNH patients evaluated in six international reference PNH centers (Paris, Naples, London, Florence, São Paulo, and Ribeirão Preto). Results Forty-two patients were male and 51 were female; the median age at diagnosis (treatment starting) was 36 years (range, 12 to 74 years). All the patients have started Ecu treatment for hemolytic PNH; 57% of patients had classic PNH, whereas 30% and 13% had aplastic anemia (AA)/PNH or intermediate PNH (Peffault de Latour et al, Blood 2006), respectively. Two-thirds of patients had been receiving red blood cell (RBC) transfusion before starting Ecu; the mean treatment duration was 97 months (range 6-179). The majority of patients (87%) received Ecu at the standard schedule of 900 mg every 2 weeks; due to chronic, residual, intravascular hemolysis 13% were treated with higher doses (1200 mg) or with shorter administration interval (10-12 days). Hematological responses were evaluated early during the treatment course (at 6 and 12 months), and then later during the last 6 months of treatment (Figure 1). After 6 (n=80) and 12 months (n=79) of Ecu, respectively, rates of hematological response were as follows: complete response 9% and 10%, major response 4% and 6%, good response 34% and 47%, partial response 34% and 28%, minor response 14% and 6% and no response 6% and 3%. In 83 patients treated for more than 18 months late response rates also were evaluated; most patients responded and the rates of hematological response during the last 6 months of treatment were as follows: complete response, 11%; major response, 6%; good response, 41%; partial response, 29%; minor response, 7% and no response, 6%. Breakthrough episodes were recorded in 21% of patients (equally split between pharmacokinetic and pharmacodynamic ones); thromboembolic events and AA were seen in 2% and 3% of patients, respectively. These data suggest that, albeit individual and unpredictable, the extent of the hematological benefit of Ecu in most PNH patient can be determined quite early in the treatment course, after a minimum of 12 months. Conclusion This is the first attempt to classify hematological response to Ecu based on Hb levels and residual hemolysis. The proposed classification allows the identification of distinct patient subgroups with different medical needs, as well as of meaningful clinical goals for future anti-complement therapies. In our cohort, about 60% of patients are well controlled with Ecu, even if less than 20% of patients reach normal Hb values; about 40% of patients remain anemic, and this represents a goal for future therapies in PNH. This classification is helpful to characterize more precisely the clinical response, which should be assessed after 1 year of treatment. Even if it should be validated in independent, larger cohorts, this classification represents a useful tool to design future trials, and to compare the clinical results of the novel anti-complement agents in development for PNH. Disclosures Notaro: Alexion: Membership on an entity's Board of Directors or advisory committees, Other: letture fees. Scheinberg:Pfizer,: Speakers Bureau; Celgene: Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Alexion: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Kulasekararaj:Alexion: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene Corporation: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Achilleon: Consultancy; Ra Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; Akari Therapeutics: Consultancy. Risitano:Biocryst: Membership on an entity's Board of Directors or advisory committees; Samsung: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Ra Pharma: Research Funding; Samsung: Membership on an entity's Board of Directors or advisory committees; Achillion: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Apellis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Alexion: Honoraria, Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Ra Pharma: Research Funding; Alnylam: Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Alnylam: Research Funding; Amyndas: Consultancy; Achillion: Research Funding; Amyndas: Consultancy; Biocryst: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Apellis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Alexion: Honoraria, Research Funding, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees. Peffault de Latour:Pfizer: Consultancy, Honoraria, Research Funding; Alexion: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Amgen: Research Funding.

Abstract: Single-agent monoclonal antibodies targeting the immune checkpoint PD1 (programmed death 1) are an efficient and safe therapeutic option in patients with relapsed/refractory B-cell lymphoma. However, many patients progress or lose response to anti-PD1. Recent studies have highlighted the role of the gut microbiota (GM) in influencing the response to chemo-immunotherapeutic agents. Here we hypothesize that the GM dynamics in B-cell lymphoma patients during anti-PD1 therapy correlate with treatment response. From December 2017 to December 2020, we enrolled 17 patients (12 with classical Hodgkin lymphoma [cHL] and 5 with primary mediastinal B-cell lymphoma [PMBCL] ) treated with anti-PD1 due to relapsed/refractory disease. Feces were collected at baseline, before each therapy cycle, at response assessment (both during therapeutic course and at the end of treatment) and for grade & gt;2 adverse events. All the samples were profiled through Illumina sequencing. At each time point, patients compiled a 7-day weighted food intake record that was analyzed by MètaDieta (METEDA). We report the results of the first 6 patients enrolled, all affected by cHL. Of the six patients, five were females. The median age was 31 years (range 26-71). Five patients were refractory to the last therapy, with a median of previous treatments of 3 (range 3-5). All the six patients discontinued the chemo-immunotherapy. In particular, three patients were discontinued due to disease progression, two achieved a complete remission and consolidated the response with autologous stem cell transplantation and the last one discontinued due to a grade 3 adverse event, despite partial remission. The median number of anti-PD1 cycles was 15 (range 7-18). The baseline GM was found to be distinct from that of age-/gender-matched healthy controls (HC). In particular, the Bray-Curtis dissimilarity index showed significant segregation between the two study groups (p value & lt; 1×10 -4, PERMANOVA), as well as greater dispersion in the patient group. Regarding intra-individual diversity, although no significant differences were found with both metrics used (Inverse Simpson and Shannon index, Wilcoxon test), a slight decrease in patients compared to HC was observed. By analyzing the genus-level composition, compared to HC, the microbial ecosystem of patients was enriched in the pathobiont Collinsella while depleted of health-associated taxa, e.g., Faecalibacterium, Ruminococcus, Coprococcus and Roseburia (p value & lt; 0.05; Figure 1A). When focusing the analysis on the GM trajectories along the checkpoint inhibitor treatment (Figure 1B), we found that intra-individual variability underwent cyclical fluctuations in responders, while values remained nearly constant in non-responders. Furthermore, significant differences were found between the GM structures of the two patient groups (responders vs non-responders), both at the level of dominant (weighted UniFrac, p value = 0.02) and subdominant (unweighted UniFrac, p value = 0.01) microbial components. The analysis of the questionnaires on eating habits filled in by the patients at each time point made it possible to estimate the daily consumption of the main macronutrients. Despite comparable energy intake and protein and fiber consumption, a greater lipid consumption and lower carbohydrate consumption were observed in responders than in non-responders. In conclusion, the GM of patients affected by B-cell Hodgkin lymphoma showed some peculiarities compared to the HC microbiota, with a depletion of health-promoting microbial components, including producers of short-chain fatty acids (i.e., Faecalibacterium, Roseburia, Coprococcus and Ruminococcus). During therapy, a peculiar trend of GM response emerged in patients with different therapeutic outcomes. Beyond the different structures in terms of dominant and subdominant microbial components, responders showed greater biodiversity plasticity than non-responders. This difference possibly suggests a lower resilience of the disease state. Furthermore, the responder group showed a greater consumption of lipids and a lower intake of carbohydrates during therapy, opening interesting perspectives towards the development of integrated intervention strategies in this peculiar setting. Figure 1 Figure 1. Disclosures Zinzani: GILEAD: Other: Advisory board, Speakers Bureau; MSD: Consultancy, Other: Advisory board, Speakers Bureau; TAKEDA: Other: Advisory board, Speakers Bureau; TG Therapeutics: Other: Advisory board, Speakers Bureau; BMS: Other: Advisory board, Speakers Bureau; Beigene: Other, Speakers Bureau; Incyte: Other, Speakers Bureau; SANDOZ: Other: Advisory board; ADC Therap.: Other; JANSSEN-CILAG: Other: Advisory board, Speakers Bureau; CELLTRION: Other: Advisory board, Speakers Bureau; EUSAPHARMA: Consultancy, Other, Speakers Bureau; KYOWA KIRIN: Other, Speakers Bureau; NOVARTIS: Consultancy, Other, Speakers Bureau; SERVIER: Other: Advisory board, Speakers Bureau; ROCHE: Other, Speakers Bureau; VERASTEM: Consultancy, Other: Advisory board, Speakers Bureau.

Abstract: Purpose: Lonidamine (LND) can enhance the activity of anthracyclines in patients with metastatic breast cancer. A multicenter, prospective, randomized trial was designed to determine whether the association of LND with high-dose epirubicin plus cyclophosphamide (EC) could improve disease-free survival (DFS) in patients with early breast cancer (BC) compared with EC alone. Granulocyte colony-stimulating factor (G-CSF) was added to maintain the EC dose-intensity. Patients and Methods: From October 1991 to April 1994, 506 patients with stage I/II BC were randomly assigned to four groups: (A) epirubicin 120 mg/m 2 and cyclophosphamide 600 mg/m 2 administered intravenously on day 1 every 21 days for four cycles (124 patients); (B) EC plus LND 450 mg/d administered orally (125 patients); (C) EC plus G-CSF administered subcutaneously (129 patients); (D) EC plus LND plus G-CSF (128 patients). Results: Median follow-up was 55 months. Five-year DFS rate was similar for LND (B+D groups; 69.6%) versus non-LND arms (A+C groups; 70.3%) and G-CSF (C+D groups; 67.2%) versus non–G-CSF arms (A+B groups; 72.9%). Five-year overall survival (OS) was comparable in LND (79.1%) versus non-LND arms (81.3%) and in G-CSF (80.6%) versus non–G-CSF arms (79.6%). DFS and OS distributions in LND and G-CSF arms did not change according to tumor size, node, receptor, and menopausal status. G-CSF dramatically reduced hematologic toxicity without having a significant impact on dose-intensity (98.1% v 95.5% for C+D and A+B groups, respectively). Conclusion: EC is active and well tolerated in patients with early breast cancer. The addition of LND or G-CSF does not improve DFS or OS.

Abstract: e12043 Background: Significant and symptomatic cardiac comorbidity (CC) is a contraindication to adjuvant trastuzumab (T) in breast cancer patients (pts). However, some pts with asymptomatic, non-limiting CC and normal baseline left ventricular ejection fraction (LVEF) receive T in the clinical practice. We sought to describe the tolerability of T in these pts. Methods: Retrospective analysis of pts with baseline asymptomatic non-limiting CC receiving adjuvant T with chemotherapy (CT) at 6 Institutions between Jul 2006 and Jan 2016. Results: Thirty-seven patients HER2-positive, operable BC at high risk of relapse BC were studied. Median age was 64y (range 36-82, 80% post-menopausal), median baseline LVEF was 61% (range 50-85%) and median BMI 26 (18-42, obesity 22%). Thirteen patients (35%) received T with adjuvant anthracycline and taxane-based, 19 (51%) taxane-based and 3 (8%) other adjuvant CT regimens (13 pts sequential, 22 pts concomitant with CT) and 2 (5%) with endocrine therapy. Prior non-limiting CC was ischemic heart disease (35%), valvular disease (30%), atrial fibrillation (19%), conduction disorders (13%), aortic aneurism (3%), and other (19%). Nine (29%) pts experienced TRC: congestive heart failure (1 pt, 3%), LVEF reduction (6 pts, 16%) and rhythm disturbances (1 pt, 3%). TRC occurred in pts with ongoing multiple cardiovascular risk factors (i.e. obesity and hypertension). Seven pts discontinued T because of TRC (19%), 5 permanently (14%) and 2 temporarily (5%). These latter pts, were able to resume and complete T after TRC resolution. At the end of adjuvant treatment, all pts showed LVEF within normal limits, except one of those who experienced a TRC (last FU value 46 %). Conclusions: This is the first analysis of TRC in pts receiving adjuvant T in the presence prior non-limiting CC. Despite the small size, our analysis shows that T is feasible in these pts and most of the TRC events were reversible at T withdrawal. Caution is needed in pts with significant ongoing cardiovascular risk factors, but when adjuvant T is deemed beneficial on breast cancer outcomes, non-limiting CC should not preclude treatment.

Abstract: 11 Background: Differences in fixation and IHC and subjective interpretation can substantially affect the accuracy and reproducibility of estrogen receptor (ER), progesterone receptor (PR) and HER2 expression. The commercially available TargetPrint test measures the mRNA expression level of ER, PR and HER2 and is 98% concordant with centrally assessed ER as presented by Viale et al, SABCS 2011. This study compares results from the microarray-based TargetPrint with IHC and FISH (for HER2 IHC2+) generated by local standard procedures. Methods: Fresh tumor samples (core needle biopsies or surgical) were collected for 831 patients diagnosed with breast cancer stage I to IV (Feb 2008 - Jan 2011) from 22 hospitals from Europe, New Zealand, Japan and US. The results of the IHC/FISH assessments performed according to the local standards at the hospitals were compared to the quantitative gene expression readouts with TargetPrint. Discordant cases were centrally reviewed for IHC/FISH assessment. Results: Of the 831 samples, IHC assessment was unknown for 4 ER/ PR samples; HER2 was unknown for 12 samples. Comparison of IHC and gene expression read out by TargetPrint showed a concordance of 95% for ER; 83% for PR and 94% for HER2. In this study, 3% of all IHC ER positive samples were classified negative by microarray, and 11% of IHC PR positive samples were classified negative by microarray. For HER2, 4% of IHC/FISH HER2 positive samples were classified negative by microarray and 2% of IHC/FISH HER2 negative samples were classified positive by microarray. Most notably, all available 5 ER IHC negative/TargetPrint positive samples turned out to be positive with central re-assessment. HER2 IHC2+ samples with discordant classifications for TargetPrint and local assessment are currently being reviewed for FISH/SISH assessment. Conclusions: Microarray based readout of ER, PR and HER2 status using TargetPrint is fairly comparable to local IHC and FISH analysis in 827 analyzed samples in various hospitals worldwide. However, re-assessment of discordant cases–especially IHC ER-/TargetPrint ER+ cases- confirms TargetPrint to be a useful high quality second opinion for local IHC/FISH assessment.