ALBERT

Description: Introduction The TP53 gene encodes the tumour suppressor and cell cycle regulatory protein and is found to be mutated in a variety of carcinomas. Mutation in TP53 gene is associated with resistance to conventional therapy, disease progression and overall poor prognosis in solid tumours and haematological malignancies including Myelodysplastic Syndromes (MDS). TP53 mutated sub-clones in MDS have been demonstrated by deep sequencing technology in prior studies. Strong nuclear staining of p53 protein by immunohistochemistry has been used as a surrogate marker for TP53 gene mutation in haematological and other malignancies. Methods We analysed sequential marrow samples for p53 expression on 35 patients with MDS from a single institution pre and post Azacitidine therapy. Formalin fixed, paraffin embedded marrow biopsies were stained with DO-7 mouse p53 monoclonal antibody. 1000 haematopoietic cells were examined under the high power and p53 expression was determined as per Modified Quick Score. Results Median age of the patients was 70 and WHO subgroups were identified as follows: 7 RCMD, 1 5q-syndrome, 1 MDS/MPN, 8 CMML, 6 RAEB-1, 6 RAEB-2 and 6 t-MDS. Cytogenetic risk as per IPSS-R/CPSS showed 17 (50%) lower risk, 4(12%) intermediate risk and 13(38%) higher risk groups. Patients received a median 13 cycles of Azacitidine. Marrows were assessed prior to treatment and after 3-6 cycles. Median overall survival of the study group was 20 months and transformation to AML occurred in 13 patients (37%). At diagnosis, 27 patients (77%) were p53 negative and 8 patients (23%) were p53 positive. At reassessment, 24 patients (69%) remained p53 negative while 6 patients (17%) remained p53 positive. Two patients (6%) became p53 negative and 3 (8%) became p53 positive following Azacitidine treatment. Median overall survival of patients who remained p53 negative during Azacitidine treatment was 28 months compared to 11 months in patients who remained p53 positive, p=0.005. Similarly, median overall survival of patients who remained or became p53 negative was 28 months compared to 18 months for those who remained or became p53 positive during Azacitidine therapy, p=0.012. p53 expression at diagnosis or changes in p53 expression during Azacitidine treatment did not correlate with transformation to Acute Myeloid Leukaemia (AML) or time to progression to AML. Among the p53 positive group, patients who had more than 10% p53 expression had lower overall survival compared to those who were 10% at DiagnosisPositive p53

Description: Background: Approximately 40% of AML patients with IDH1 mutation respond to monotherapy with the IDH1 inhibitor ivosidenib with a median duration of response of 8.2 months, suggesting that IDH1 inhibitors should be rationally combined with other agents to improve efficacy. We have previously shown the synergistic activity of the mutant IDH1 (mIDH1) inhibitor BAY1436032 with azacitidine. We have also shown that the leukemogenic activity of the mIDH1 protein depends on PHD3 independent of R-2HG and its inhibition as a novel therapeutic strategy (Chaturvedi et al. 2018). Inhibition of Brd4 has been shown to induce rapid differentiation and death of IDH2 mutated AML mouse models (Chen et al., 2013). In the present study, we assessed the combination of either conventional chemotherapy, prolyl hydroxylase (PHD) inhibitor molidustat or bromodomain inhibitor JQ1 with BAY1436032 (BAY) in a preclinical patient-derived xenograft (PDX) model of mIDH1 AML. Methods and Results: Leukemic cells from an AML patient with mutated IDH1, NPM1, FLT3-TKD and NRAS were xenografted in immunocompromised mice. We investigated the effects of BAY in sequential (seq) or simultaneous (sim) combination with cytarabine plus doxorubicin in our mIDH1 PDX model. The control groups were treated with either vehicle, BAY (150 mg/kg once daily p.o. continuously), or chemotherapy, which consisted of cytarabine (50 mg/kg once daily days 1-5 i.v.) and doxorubicin (1 mg/kg once daily days 1-3 i.v.). The treatment was repeated once after 29 days. The test groups were treated with BAY and chemotherapy in the doses mentioned above either starting both drugs on day 1 (sim group) or starting chemotherapy on day 1 and BAY on day 6 (seq group). Treatment with BAY was stopped after 12 weeks. Leukemic cells in peripheral blood constantly increased in vehicle and chemotherapy-treated mice with median time to 50% engraftment (MT 50%) of 84 and 112 days respectively. After stop of treatment, the percentage of leukemic cells increased in the group receiving BAY1436032 (MT 50%: 252 days) and sequential combination (MT 50%: 280 days), however, the MT50% was not reached with the simultaneous treatment (P

Description: Background: The presence of significant marrow fibrosis has previously been shown to be a poor prognostic factor in patients with myelodysplastic syndrome (MDS). Associations between fibrosis and higher transfusion requirements, multilineage dysplasia, and an increased rate of leukaemic transformation have been demonstrated. Currently, the presence of fibrosis is not included in standard risk scores for MDS such as the Revised International Prognostic Scoring System (IPSS-R) nor is fibrosis included in the current WHO classification for MDS. It is also not certain whether the presence of fibrosis should alter current treatment algorithms for patients. More information is needed to further assess both the prognostic and therapeutic implications of the presence of significant fibrosis in this patient population. Methods: We conducted a retrospective study utilizing a database of 247 patients with diagnosed MDS in a single center from 2000-2014. Bone marrow trephine samples for 200 of these patients were assessed using the European consensus on grading bone marrow fibrosis. Data was collected on: age, gender, WHO classification, marrow blast percentage, cytogenetics, progression to AML, treatment with azacitadine, and overall survival. These characteristics were compared between patients without significant fibrosis (grade 0/1) and those with significant fibrosis (grade 2/3). Our aim was to identify potential significant associations with MDS fibrosis and to assess whether treatment with azacitadine influenced these parameters or overall survival between the two groups. Results: Of 200 patients, 38 were found to have significant fibrosis (19%) versus 162 without significant fibrosis (81%). There was no significant difference in age or gender between the two groups. The commonest WHO category in both groups was RCMD (31.58% v 48.77%) in the fibrosis and non-fibrosis groups respectively. IPSS-R score was determined for 152 patients where data was available. There was no significant difference observed in IPSS-R Score between the two groups. Cytogenetic data was available on 175 patients. The commonest cytogenetic result in both groups was normal karyotype (55.3% v 52.5%). In the fibrosis group this was followed by complex cytogenetics (〉 2 abnormalities) (23.68%) and trisomy 8 (7.89%). The presence of a cytogenetic abnormality was not significantly different in those with or without fibrosis (p=0.69). A significant difference was found between patients' marrow blast percentage at diagnosis (average 5.4% blasts in fibrotic patients versus 3.6% in non-fibrotic patients (p=0.04)). There were no differences in diagnostic haemoglobin level, neutrophil count, and platelet count. Acute Myeloid Leukaemia (AML) developed in 31 patients. The presence of fibrosis was associated with an increased rate of AML transformation with 27% compared with 13.5% in patients without fibrosis (p=0.05). Median overall survival was decreased in the fibrosis group compared to the non-fibrosis group (29 months versus 42 months, p=0.02). A total of 36 patients (25 of whom progressed to AML) received azacitadine treatment (9 (24%) patients with fibrosis and 27 (17%) patients without fibrosis). Patients with fibrosis had a longer median survival than those without (29 months and 19 months respectively) but this difference was not statistically significant (p=0.48). Conclusion: Marrow fibrosis adversely affects overall survival in patients with MDS. Patients with fibrosis are more likely to present with higher marrow blast counts and to progress to AML. Patients with fibrosis who received azacitadine appeared to have an overall higher survival than those without fibrosis. However, the numbers of patients who received azacitadine were small. This study confirms the adverse prognostic influence of marrow fibrosis in patients with MDS, but the presence of fibrosis may not adversely affect the responsiveness to azacitadine therapy. Table IPSS-R Score - Fibrosis versus Non-Fibrosis Table. IPSS-R Score - Fibrosis versus Non-Fibrosis Figure Overall Survival (Non-Fibrosis versus Fibrosis) Figure. Overall Survival (Non-Fibrosis versus Fibrosis) Disclosures Desmond: Novartis: Honoraria.

Description: Abstract 3704 Background: BAY 80–6946 is a potent and highly selective, reversible, pan-Class I phosphatidylinositol-3-kinase (PI3K) inhibitor, with broad antitumor activity in a panel of preclinical models including both indolent and aggressive NHL. Even in the absence of PIK3CA mutations, the PI3 pathway has been demonstrated to be constitutively activated in the majority of B-cell lymphomas. BAY 80–6946 was more potent than CAL-101 in inhibiting the in vitro growth of a panel of leukemia/lymphoma cell lines. Methods: In a phase I dose escalation study, BAY 80–6946 was previously reported to be tolerated as a 1-hr infusion at a dose of 0.8 mg/kg (MTD) on days 1, 8 and 15 every 28 days (J Clin Oncol 29: 2011 suppl; abstr 3035). Additional patients were treated in MTD expansion cohorts to assess safety, PK, biomarkers and clinical benefit in selected patient populations, including one expansion cohort in NHL. Based upon the beneficial clinical responses observed in 5 follicular lymphoma (FL) patients (J Clin Oncol 30: 2012 suppl; abstr 3019), the NHL cohort was expanded up to a total of 12 patients. Samples were collected for pharmacokinetic analyses. Exploratory analyses of changes in several plasma proteins, chosen with emphasis on B-cell homing/survival, were performed. Response was assessed using International Working Group response criteria, including 18FDG-PET. Results: To date, 9 NHL patients have been enrolled (FL, n=6, DLBCL, n=3). There were 5 females/4 males with a median age of 72 years (range, 40–84). Among these 9 patients, 5 (56%) had received 3 or more prior regimens. All have received prior Rituximab and 6/9 prior anthracycline and 4/9 prior Bendamustine. Eight patients were evaluable for safety and tolerability. The most frequently drug-related adverse events reported in 〉20% of the patients were hyperglycemia, nausea, diarrhea, anemia, mucositis, and fatigue. Interstitial pneumonitis was observed in 2 patients with FL. Both patients responded to corticosteroids and one continued on treatment at full dose without recurrence of the pneumonitis. The pharmacokinetic parameters of BAY 80–6946, including the Cmax and AUC, were consistent with those seen in solid tumour patients. Changes in the plasma levels of CXCL13 and BAFF were observed in subjects treated with BAY 80–6946. Median time on study was 129 days (3–487). Best response in 6 evaluable patients (FL, n=5, DLBCL, n=1) was 5 PR and 1 PD, all 5 evaluable FL patients achieving a PR, the longest of which is 〉16 months. Pharmacodynamic effects were demonstrated with significant lymphoma shrinkage observed as early as 48 hours after the first dose of BAY 80–6946 on 18FDG-PET/CT in both FL and DLBCL patients. Conclusions: In this MTD expansion cohort study in NHL lymphoma, BAY 80–6986 was generally well tolerated and showed very promising clinical activity in NHL patients. Updated clinical, PK and pharmacodynamic results will be presented. Based on these preliminary results, further clinical development as a single agent or in combination regimens in NHL is warranted. Disclosures: Patnaik: Bayer: Research Funding. Ramanathan:Bayer: Research Funding. Appleman:Bristol-Myers: Research Funding; Bayer Pharmaceuticals: Research Funding; AstraZeneca: Research Funding; Amgen: Research Funding; Abbott: Research Funding; Cougar Biotechnology/Ortho Biotech: Research Funding; Medivation: Research Funding; Excelixis: Research Funding. Beerham:Bayer: Research Funding. Weiss:Bayer: Honoraria; Roche/Ventana: Honoraria; Merrimack: Honoraria; Cephalon: Honoraria; Eli Lilly: Honoraria; Cytrx: Consultancy; Genentech: Speakers Bureau; Pfizer: Speakers Bureau; Caris Life Sciences: Speakers Bureau; Bayer: Research Funding. Rajagopalan:Bayer Pharmaceuticals: Employment. Jeffers:Bayer Pharmaceuticals: Employment. Kelly:Bayer Pharmaceuticals: Employment. Genvresse:Bayer Pharma AG: Employment.

Description: Therapy-related myelodysplastic syndromes and acute myeloid leukaemia (t-MDS/AML) occur as a late complication of cytotoxic chemotherapy and/or radiation for neoplastic and non-neoplastic disorders and are a major cause of non-relapse mortality. The prognosis of t-MDS/AML is poor because of an increased incidence of adverse cytogenetic features and relative resistance/intolerance to conventional therapies. TP53 is a tumour suppressor and cell cycle regulatory protein. Mutations in the TP53 gene are known to be associated with adverse outcomes in a variety of cancers including haematological malignancies. We examined p53 expression using immunohistochemical staining, as a surrogate marker for TP53 mutation, in marrow biopsies of 39 patients with t-MDS/AML from a single institution and correlated p53 expression with survival. Formalin fixed, paraffin embedded marrow trephine samples at diagnosis of t-MDS/AML were stained with DO-7 mouse p53 monoclonal antibody. Positive expression was defined as per Modified Quick Score. Of 39 patients, 35 had t-MDS and 4 had t-AML. 51% of patients had marrows at diagnosis that showed p53 positivity and 49% were negative. In a control group of 47 MDS patients in our centre, 22% were p53 positive and 78% were p53 negative. Of the t-MDS/AML group, age at diagnosis and gender distribution was similar in p53 positive and negative patients. Median latency (time from diagnosis of primary malignancy to diagnosis of t-MDS/AML) was similar between p53 positive and negative patients (66 vs. 52 months, p=0.51). A similar distribution was noted for the type of primary tumours (haematological vs. solid tumour) among the two groups, but all patients with more than one prior malignancy were noted to be p53 positive (N=4) whereas all patients who developed t-MDS after cytotoxic therapy for non-neoplastic disorders were p53 negative(N=3). There was no difference in p53 expression based on the type of primary therapy received (chemotherapy and/or radiotherapy) but a greater proportion (71%) of patients who received combined chemotherapy and radiation was p53 positive. A higher proportion of p53 negative patients had lower risk cytogenetics and normal karyotype whereas in p53 positive group there was lower incidence of good risk and a higher proportion to intermediate risk cytogenetics. However, equal numbers of patients with higher risk cytogenetics were found between p53 positive and negative groups. These findings were again reflected by IPSS-R risk groups (Table 1). Median overall survival was 10.5 months in p53 positive group compared to 22.5 months in p53 negative patients, p= 0.208. (Figure 1) In conclusion, our study demonstrated that a higher proportion of patients with t-MDS/AML were positive for p53 than in de novo MDS which may be related to poor outcomes in this group. p53 positive t-MDS/AML patients tend to have lower incidence of favourable karyotypes and showed poor survival compared to their counterparts who were p53 negative. Analysis of p53 expression by immunohistochemistry is a readily accessible, cost-effective method of assessment without the need for expensive gene sequencing and is a clinically useful prognostic tool. It may be particularly useful in patients with t-MDS/AML. Table 1. p53 expression in Therapy-related Myeloid Neoplasms p53 Positive (N=20, 51%) p53 Negative (N=19, 49%) Primary TumourHaematological (22) Solid Tumour (10) Autoimmune disease (3) More than one disorder (4) 45% 60% 0 100% 55% 40% 100% 0 Primary TherapyChemotherapy alone (27) Radiotherapy alone (5) Combined therapy (7) 44% 60% 71% 56% 40% 29% Cytogenetic RisksLow (very good, good) (14) Intermediate (8) High (poor, very poor) (12) 29% 75% 50% 71% 25% 50% IPSS-RLower (very low, low)(12) Intermediate (4) Higher (high, very high) (16) 33% 75% 50% 67% 25% 50% Disclosures No relevant conflicts of interest to declare.

Description: Histone deacetylase (HDAC) inhibitors represent a new mechanistic class of anti-cancer therapeutics that inhibit HDAC enzymes and have been shown to have anti-proliferative effects in cancer cells (including drug resistance subtypes), induce apoptosis, inhibit angiogenesis, and sensitize cancer cells when combined with other available anti-cancer therapies. PXD101 is a novel investigational small molecule drug that selectively inhibits HDAC enzymes. In recent preclinical studies, PXD101 has been shown to have the potential to treat a wide range of solid and hematological malignancies either as a monotherapy or in combination with other active agents. In this study, we evaluated the activity of PXD101 on multiple myeloma samples when used as monotherapy or in combination with the proteasome inhibitor bortezomib. In vitro experiments indicated that PXD101 pretreatment (20 mM; 3h) sensitized RPMI-8226 human multiple myeloma cells to subsequent bortezomib exposure (5 nM; 72h). To examine PXD101 and bortezomib in vivo, two mouse models of human multiple myeloma were utilized (LAGλ-1 and LAGκ-1B). LAGλ-1 was generated from a patient resistant to melphalan therapy and LAGκ-1B from a patient who progressed on bortezomib treatment (Campbell et al, International Journal of Oncology 2006). SCID mice were implanted with LAGλ-1 or LAGκ-1B tumor fragments into the left superficial gluteal muscle. Tumors were allowed to grow for 14 days at which time human IgG levels were detectable in the mouse serum, and mice were randomly assigned into treatment groups. Groups consisted of Vehicle only, PXD101 alone (40 mg/kg), bortezomib alone (0.5 mg/kg), or PXD101 (40 mg/kg) + bortezomib (0.5 mg/kg). In one cohort, PXD101 and bortezomib were administered twice weekly (M, Th) and in another cohort PXD101 was administered 5 days a week (M-F) and bortezomib twice weekly (M, Th). When administered, PXD101 was given i.p twice daily and bortezomib once daily intravenously. The results of these animal experiments will provide preclinical information on the activity of PXD101 monotherapy and PXD101/bortezomib combination therapy on drug-resistant myeloma samples, and may help to define the optimal schedule for potential clinical evaluation of this drug combination.

Description: Previous investigations have shown that the novel hydroxamate PXD101 potently inhibits the enzymatic activity of histone deacetylase (HDAC) at nanomolar concentration, and the growth of cancer cell lines derived from solid malignancies at low micromolar potency. We have now examined and present results showing the activity of PXD101 on 〉 20 cell lines corresponding to a variety of haematological cancer types including acute T-cell leukaemia, cutaneous T-cell lymphoma, anaplastic large T-cell lymphoma, mantle cell lymphoma, Burkitt’s lymphoma, diffuse large-cell lymphoma, plasma cell leukaemia and multiple myeloma (MM). Our data show that PXD101 strongly inhibits the growth of these cancer types at submicromolar potency. In addition, we will present in vitro data demonstrating that PXD101 efficiently inhibits the growth of doxorubicin-resistant leukemic cells, thereby supporting the possibility that this compound may be effective on drug-resistant haematological cancers. Finally, we will present results demonstrating that PXD101 used in combination with Velcade mediates greater growth-inhibition than does either drug used as monotherapy on a multiple myeloma cell line. These promising preclinical findings, taken together with previous results demonstrating the activity of PXD101 in a murine leukaemia animal model, support the evaluation of PXD101 for the treatment of haematological malignancies in a clinical setting. To this end, a Phase I trial using PXD101 in patients with advanced hematological tumors refractory to standard therapy was initiated. PXD101 is administered as a 30 minute i.v. infusion on days 1 to 5 of a 21 day cycle. Dose levels 600 mg/m2/d and 900 mg/m2/d have been examined (3 patients each) and recruitment to the dose level 1000 mg/m2/d is ongoing (5 patients to date). Patient characteristics are 8 males and 3 females with median age of 67 (range 58 – 73). Median number of prior therapies is 5 (range 4 – 7). Of the 11 patients enrolled to date, diagnoses were: MM (2), Non-Hodgkin’s lymphoma (5), CLL (4). Eleven patients have been treated with 1–8 cycles of therapy. Adverse events related to PXD101 treatment have mainly been grade 1–2. The most frequent adverse events were nausea (12/23 cycles), fatigue (9/23 treatment cycles), vomiting (8/23 cycles) and diarrhoea (7/23 cycles). Potential antineoplastic activity has been observed. One patient with MM had a possible tumor lysis syndrome with decrease in M-component, increasing urate and serum creatinine requiring dialysis. In addition, one patient with Richter transformation of CLL refractory to other treatments has now been in stable disease for 〉 6 months. We continue to accrue patients in this study, and updated data will be presented.