ALBERT

Description: Background: Single nucleotide polymorphism (SNP) and comparative genomic hybridization (CGH) microarray analysis is a powerful tool to assess myelodysplastic bone marrow specimens for the presence of genomic gains and losses as well as loss of heterozygosity (LOH) (reviewed by Nybakken & Bagg, JMD 2014). Its application can be a valuable addition to conventional cytogenetic analysis and may be superior to FISH testing for MDS assessment. Currently, microarray analysis does not have widespread use in an MDS work-up. Several groups have demonstrated that flow cytometric analysis can detect phenotypic aberrations in bone marrow aspirates with cytopenias with more abnormalities identified in patients with poor prognosis or with multiple genotypic abnormalities (Loken et al. 2008; Cutler et al. 2011; van de Loosdrecht et al. 2013). In this study SNP microarray results were compared with conventional cytogenetic and MDS panel FISH findings as well as phenotypic abnormalities detected by flow cytometry. Patients and Methods: 185 bone marrow aspirate specimens submitted to our laboratory for MDS work-up were analyzed by SNP/CGH studies. 36 of these (19.5%) were positive by SNP/CGH microarray analysis. 32 of the positive microarray cases (88.9%) were also analyzed by conventional cytogenetic studies, 35 (97.2%) by MDS FISH panel (5p, 7q, +8, -17p, -20q) and 31 (86.1%) were assessed by multidimensional flow cytometry (FCM) and were assigned an FCSS score (Wells et al. 2003). Results: Of the specimens in which the SNP/CGH array demonstrated genotypic abnormalities, 11/32 (34.4%) were negative by conventional cytogenetic analysis while 12/35 (34.3%) showed no abnormalities by MDS FISH panel analysis. SNP/CGH analysis revealed additional chromosomal gains and losses in 18/32 (56%) in comparison to cytogenetic analysis and in 22/35 (63%) in comparison to FISH analysis. Loss of Heterozygosity regions were detected in 28/36 cases (78%) with 96.4% (27/28) of these being larger than 2 Mb and 53% (19/28) spanning a significant chromosomal region (e.g. 1p, 5q, 7q and 17p) with known oncogenic and other MDS related genes. In 10/32 cases (31%), microarray analysis was able to characterize the origin of marker chromosome material, previously reported with unknown identity by conventional cytogenetic analysis. In an additional subset of 10 out of 32 cases (31%), cytogenetic analysis was able to either characterize balanced translocations or low level sub-clonal abnormalities not identified by microarray analysis alone. In 11/36 (31%) microarray analysis was able to detect clonal heterogeneity and evolution. In none of the specimens did FISH analysis detected abnormalities not revealed by microarray analysis. Flow cytometry performed on 31 of the array positive specimens revealed 6 to have 〉20% abnormal myeloid progenitor cells (classified as AML) while 23 the remaining 25 cases showed phenotypic abnormalities consistent with MDS (FCSS ranging from 1-6). In two specimens with a FCSS of 0, LOH regions on 16q or 1p and 21q were found, respectively, without the presence of numerical aberrations. A FCSS score of 1 with minimal phenotypic abnormalities (n=3), was comprised of one specimen with del(5q), one with LOH of 7q and one with trisomy 8, 1p loss and 1q gain. Specimens with an FCSS of 2 (n=7) showed only one specimen classified as complex (5 or more abnormalities). The two FCSS =3 specimens showed del(5q) with del(12p) and several LOH regions, not complex findings. One of the 4 specimen with FCSS = 4 was classified as complex while the other 3 specimens showed monosomy 7, LOH of 7q or LOH of 1p, respectively. Genotypic abnormalities were also related to phenotypic abnormalities in 4/7 (57%) specimens in the FCSS = 5/6 category which revealed complex microarray findings. Half (3/6) of the AML class had complex findings as well. Conclusions: These results emphasize the additional value that CGH/SNP microarray analysis adds to conventional cytogenetic analysis. Our dataset confirms that FISH studies do not provide additional information for MDS specimens positive by cytogenetic and/or microarray analysis. Most importantly, a high correlation between our phenotypic flow cytometric scoring system for myeloid abnormalities and microarray findings has been identified. Higher flow cytometric abnormality scores correlate with increasing complexity of genomic abnormalities. Disclosures Zehentner: HematoLogics Inc.: Employment, Equity Ownership. Brodersen:Hematologics Inc.: Employment. Stephenson:Hematologics Inc.: Employment. de Baca:Hematologics Inc.: Employment. Menssen:Hematologics Inc.: Employment. Hammock:Hematologics Inc.: Employment. Johnson:Hematologics Inc.: Employment. Hartmann:Hematologics Inc.: Employment. Loken:Hematologics Inc.: Employment, Equity Ownership. Wells:HematoLogics Inc.: Employment, Equity Ownership.

Description: Cytogenetics has proven an essential tool not only for confirming a diagnosis/classification, but for providing prognostic value as well in myelodysplastic syndromes (MDS). However, approximately 50% of primary MDS do not show discernible chromosome changes. In recent years, the fluorescence in situ hybridization (FISH) technique using gene or chromosome locus/region specific probes has emerged as a promising test in various hematopoietic and lymphoid neoplasms. To evaluate the application of FISH panels and cytogenetic studies in MDS, we retrospectively analyzed 1,885 consecutive bone marrow results from patients with suspected MDS due to cytopenia(s). In particular, we assessed the additional information a FISH reflex testing might have given in cytogenetically normal cases. The probes used in the panel included the EGR1 at 5q31, the D7S522 at 7q31, the D8Z2 for the centromere of chromosome 8, the MLL at 11q23 and the D20S108 at 20q12 (Vysis, Inc.). Among all patients, 190 (10%) had clonal chromosome abnormalities, mostly as reported in the literatures, eg, -5/5q- accounted for 34.7% of abnormalities, trisomy 8 29.5%, -7/7q- 14.2%, 20q- 13.7%. Of 345 cases with a FISH reflex test ordered and performed, only 3 (0.87%) showed positive results: a deletion of 7q31, a deletion of 20q12 and a deletion of 5q31 in 9.6%, 8.2% and 71.5% of interphase cells respectively. For the case with 5q- detected by FISH, only 12 metaphases were available for cytogenetic analysis. From our data and experience, at present, interphase FISH panel testing seems not to be an efficient and cost-effective method used as a screening test for cytopenia(s) in the diagnosis of MDS, different from its applications in B-cell chronic lymphoid leukemias, non-Hodgkin lymphomas and plasma cell neoplasms where neoplastic cells inherited not to divide easily in culture for metaphase analysis. Rather, it should be used for suspected MDS cases as a technique of choice for problematic specimens compromised for cytogenetic analysis such as cellular insufficiency, extended transit time and extremely low mitotic index or poor chromosome morphology. Until more genetic defect targeted probes become available with a better understanding of the stem cell biology and pathogenesis in MDS, cytogenetics is still the best and a “must” technique for detecting genomic aberrations in MDS and nearly all other myeloid hematopoietic neoplastic disorders.

Description: Abstract 3933 Background Ofatumumab is a novel fully humanized anti-CD20 monoclonal antibody with antigenic target distinct from rituximab and enhanced antibody dependent cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) with single agent activity in relapsed or refractory (R/R) chronic lymphocytic leukemia (CLL), including heavily pretreated patients. The immunomodulatory agent (IMID) lenalidomide has been shown to induce T cell and NK cell activation and in vitro enhances rituximab induced killing of B-CLL cells via NK cell-mediated and monocyte-mediated ADCC. We hypothesized that the sequential treatment of patients with ofatumumab and lenalidomide would provide optimal pharmacodynamic interaction and be active in R/R patients previously treated with rituximab containing regimens. Methods Eligibility criteria included confirmed diagnosis of R/R CLL meeting criteria for treatment, prior therapy containing rituximab, age≥ 18 years, ECOG performance status ≤ 2 adequate hepatic, renal and bone marrow function and willingness to comply with the required birth control measures. Patients were excluded if they had been previously exposed to any of the experimental agents, had active hepatitis B or carried HIV. Treatment consisted of ofatumumab 2000 mg (300 mg on the first cycle) intravenously on day 1 and lenalidomide 10 mg (5mg on the first cycle) on days 8–28. Treatment was administered for up to 6 cycles of 28 days duration. Patients received prophylaxis with acyclovir and trimethoprim + sulfamethoxazole. Toxicity was assessed according to CTC v.4.0 and response was evaluated following the 2008 National Cancer Institute Working Group criteria. Results Seventeen patients have been enrolled and 14 have sufficient follow-up to be assessed for response. Median age of patients was 65 years (range 51–80). Median number of prior lines of therapy was 2 (range 1–4) and median baseline white blood cell count was 75,000/mm3. The proportions of cases with unmutated IgvH chain and positive ZAP-70 expression were 15/17 (88%) and 13/16 (81%), respectively. There were 4/16 (25%) cases with del17p and 4/16 (25%) with del11q detected by fluorescence in situ hybridization (FISH). Thirteen cases (76%) were refractory to, or had relapsed after treatment containing a purine analogue. The most frequent adverse event (AE) 〉 Grade 1 was tumor flare reaction (TFR), seen in 8/14 (57%) patients and infusion reactions seen in 6/14 (43%) patients. Four patients with TFR were managed successfully with non-steroidal anti inflammatory agents while 4 required glucocorticoids allowing continuation of therapy in all patients. The most common Grade 3+4 AE was neutropenia (11/14, 79%) although it was associated with infection in only 1 episode. One subject had early discontinuation due to toxicity (elevation in AST and ALT precluding further administration of ofatumumab). The majority of patients (11/14, 79%) required dose reduction or could not have the planned dose increase of lenalidomide after cycle 1 due to hematological toxicity. Overall 6/14 (43%) had objective response and 3/14 (21%) had stable disease for an overall clinical benefit in 64% of patients. All patients with TFR〉 Grade 1 had at least stable disease. Conclusion Intracycle sequential ofatumumab plus lenalidomide is well tolerated in advanced, high-risk CLL except for high rate of TFR and neutropenia without infection. Sequential ofatumumab and lenalidomide may be associated with higher rate of TFR than concomitant therapy. Approximately half the patients treated with this combination will obtain disease control. Further investigation is warranted in earlier lines and/or for more prolonged therapy. Disclosures: Costa: GSK: Research Funding. Off Label Use: Lenalidomide for treatment of CLL.

Description: The Multiple Myeloma Research Foundation (MMRF) CoMMpass trial (NCT0145429) is a longitudinal study of 1000 patients with newly-diagnosed multiple myeloma. The study opened July 2011 and now includes over 650 patients from 91 sites in the United States, Canada and European Union. Each patient is required to receive an approved proteasome inhibitor, immunumodulatory agent, or both. Enriched tumor and matched constitutional samples are comprehensively analyzed using Long-Insert Whole Genome Sequencing (WGS), Whole Exome Sequencing (WES) and RNA sequencing (RNAseq). Clinical parameters, Quality of Life measurements and health care resource utilization values are collected at study entry and every three months for a minimum of five years. Additional bone marrow aspirates are collected and analyzed at each recurrence or progression of disease. An extensive clinical and molecular database, the MMRF Researcher Gateway (https://research.themmrf.org), has been developed to facilitate the rapid dissemination of the results and provides the myeloma community with a mechanism to analyze the data. In this current interim analysis, we report on 195 patients that are fully characterized at the molecular level. We focused this analysis on immunoglobulin translocations and inter-chromosomal fusion transcripts. As expected we detected the classic canonical t(4;14), t(6;14), t(11;14), and t(14;16) translocations targeting FGFR3/MMSET, CCND3, CCND1, and MAF respectively. Seven patients presented with t(8;14) rearrangements correlating with high expression of MYC. Novel translocations were detected targeting MAP3K14/NIK in two patients and NFKB1, TOP1MT, TXNDC2, APOL3, FCHSD2, PRICKLE1, and BCL2L1 in individual patients. Importantly, the matched RNAseq data confirmed the high expression of MAP3K14, NFKB1, TOP1MT, APOL3 and BCL2L1. Moreover, the anti-apoptotic isoform of BCL2L1, Bcl-xL, was the prominent transcript isoform detected. In several patients we detected multiple IgH translocations. For instance the BCL2L1 translocation occurred in a downstream class switch recombination region from one associated with a co-occurring t(11:14). We also analyzed the RNAseq dataset for inter-chromosomal fusion transcripts and leveraged the independent long-insert WGS data to validate the predicted fusions. The only recurrent fusion partner identified was IgH-MMSET created by t(4:14). Fusion transcripts were detected in individual patients between IgH elements and MYEOV and WWOX along with several of the novel IgH translocation partners; NFKB1, TOP1MT, and APOL3. Several genes are involved in multiple fusions but with different partners. Three independent fusions were detected between the highly expressed gene FCHSD2 and MYC, MAP3K14, and ANKRD55. Three additional fusions were detected between MAP3K14 and ELL, PLCG2, and CDC27, which produce hybrid MAP3K14 isoforms lacking the N-terminal negative regulatory domain. We also detected three independent fusions involving BRF1, which is typically not expressed in myeloma tumors. These appear to be markers of translocations occurring just centromeric of the strong 3’ IgH enhancers. Interestingly, two of the partners are located in a region of chromosome 12 harboring MDM2 and spiked expression of MDM2 was observed. Additional genes with multiple fusion events included NEDD9 and ARHGEF12. Integrating the WES and RNAseq datasets, we identified 3518 variants (median 14 per patient) where the variant allele detected by WES, was also detected in the RNAseq data, suggesting it is potentially biologically relevant. Of these, 44 distinct genes were mutated in at least 2% of patients. The most common mutations (〉7 patients) occurred in KRAS, NRAS, IGLL5, DIS3, BRAF, ACTG1, EGR1, FAM46C, TRAF3, DUSP2, FGFR3, and PRR14L. We also identified a deletion of IKZF3/Aiolos in a patient who progressed rapidly on lenalidomide-dexamethasone. Alterations in Ikaros family members like Aiolos have recently been reported as a potential mechanism of resistance to IMiDs. As the study continues to mature, we expect it will provide unprecedented molecular characterization and correlating clinical datasets that will help define the determinants of response to anti-myeloma agents and facilitate future clinical trial designs, thus serving as a stepping-stone toward personalized medicine for myeloma patients. Disclosures Lonial: Millennium: The Takeda Oncology Company: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Onyx Pharmaceuticals: Consultancy, Research Funding.

Description: Paroxysmal Nocturnal Hemoglobinuria (PNH) is an acquired clonal stem cell disease, characterised by intravascular hemolysis, bone marrow failure and lifethreatening thromboses. The median survival is 10–15 years, with the average age of presentation being in the 30’s. Symptoms include hemoglobinuria, fatigue, anemia, venous and arterial thromboses, recurrent pain, renal impairment, erectile dysfunction and pulmonary hypertension. The care of a patient with PNH is complex and challenging, as many experience chronic symptoms with periods of acute exacerbations. Historically the management of PNH included bone marrow transplant, blood transfusion and administration of additional supportive therapies, all necessitating regular visits to the hospital. Eculizumab, a monoclonal antibody that binds to the C5 complement component inhibiting the activity of terminal complement and thus preventing the destruction of red blood cells has dramatically altered the management of hemolytic PNH. Clinical trials of eculizumab demonstrated the resolution of the majority of symptoms and complications of PNH and resulted in its approval in the UK in June 2007. Eculizumab is administered as a 30 minute intravenous infusion every 14 days, and under the terms of its current EU licence, must be administered by a healthcare professional. In view of the rarity of PNH there are relatively few specialist Centres for the disease resulting in, patients travelling long distances for review and treatment. In view of the dramatic improvement in symptoms on eculizumab many patients are able to return to a near normal lifestyle. In the UK, Leeds Teaching Hospitals with Healthcare at Home have developed a home infusion programme that ensures safe administration of eculizumab in the patient’s home at a time convenient to them, leading to enhanced treatment-associated convenience for patients and their families. Patients then only attend the PNH Centre every 3 months to ensure appropriate monitoring and patient education. A recent survey of patients reports a reduction in treatment-associated burden for PNH patients and their families when receiving infusions at home. 46 patients responded to the survey with just over half receiving eculizumab. Of the 21 patients at the time receiving home infusions 19 found this more convenient than the hospital. Home treatment allows flexibility and for some, the return to full-time employment, with the associated financial benefits and improvement in psychological well-being. Of the 21 patients on home care 7 stated there ability to work was transformed with a further 10 having great improvement. Whilst the purpose of the survey was not to address financial burden, the home infusion programme has anecdotally reduced the financial burden on the patient and their family by eliminating the need for time off work, allowing return to full-time employment, and eliminating the cost of travel to and from the hospital for treatments. No patients reporting negative impact, including effect on social life and family relationships, whilst 15 experienced improvement or complete transformation in both areas. The patients reported confidence in the homecare programme, knowing that a very close working relationship existed between the expert hospital and homecare teams. This innovative programme of medication delivery by a dedicated home nursing team allows patients who have previously struggled to cope with their illness to lead a near normal life with an associated enhancement in quality of life. Patients are able to carry on with activities of daily life, including work, recreational activities and holidays, whilst at the same time ensuring compliance with treatment and therefore allowing maximum therapeutic benefit.

Description: The RUNX1 (AML1) gene is a transcription factor that regulates expression of genes involved in hematopoietic cell differentiation. It is a gene located on chromosome 21 at q22. Genetic alterations of RUNX1 whether through loss-of-function point mutations, translocation or amplification are known to impact myeloid differentiation and trigger leukemic transformation in particular with respect to myelodysplasia and acute myeloid leukemia. However, while there are many articles describing the impact of these types of RUNX1 genetic alterations, there is a paucity of information regarding loss of the entire RUNX1 gene. The case in this abstract highlights the significance of understanding loss of the RUNX1 gene. An 87 year old patient presented for evaluation for anemia and leukopenia. Flow cytometric evaluation revealed 26% myeloid blasts and confirmed a diagnosis of acute myeloid leukemia (AML). The cytogenetic findings demonstrated a translocation between chromosomes 17 and 21 −t(17;21)(q11.2;q22). The dilemma then was to determine if this was a variant of the traditional t(15;17) associated with acute promyelocytic leukemia or a variant of the t(8;21) associated with the M2 subtype of AML. FISH studies determined that there was no involvement of the RARA gene and no evidence of a RUNX1/ETO rearrangement. However, there was a complete loss of RUNX1 on the abnormal chromosome 21. Thus, what appeared to be a balanced translocation included a cryptic loss of RUNX1. While this may be an interesting case presentation the more pertinent question is what is the impact of the RUNX1 loss? This case prompted a review of the data on complete loss of the RUNX1 gene which while limited suggests that RUNX1 loss on its own is not leukemogenic. This poster presents the data and implication of complete loss of RUNX1, the role of this loss in leukemogenesis and patient management.

Description: Human B lymphocyte-induced maturation protein-1 (BLIMP-1) was originally described as a repressor of the interferon-beta response to viral infection. Subsequently, the murine orthologue was identified as a regulator of plasma cell differentiation. The involvement of BLIMP-1 in hemopoietic differentiation is not restricted to the B-cell lineage as BLIMP-1 is induced during differentiation of myeloid progenitors. During in vitro macrophage and plasma cell differentiation the expression of BLIMP-1 is cytokine driven. However, the BLIMP-1 response to virus infection can be reproduced by transfection with double-stranded RNA (dsRNA), indicating that BLIMP-1 is a target of dsRNA responsive signaling pathways. A central regulator of the intracellular response to viral infection is the interferon-inducible double-stranded RNA activated kinase, PKR. PKR belongs to a family of kinases that phosphorylate the eukaryotic translation initiation factor 2-alpha (eIF2α) and activate common downstream signaling pathways. PERK, the endoplasmic reticulum (ER) PKR-homologue is activated during the unfolded protein response (UPR), a stress response involved in both macrophage activation and terminal B-cell differentiation. This suggested the hypothesis that BLIMP-1 may represent a shared target of signaling pathways in the response to cellular stresses such as virus infection and the UPR. In this study we demonstrate that BLIMP-1 is rapidly upregulated during the UPR in human myeloid and B-cell lines. This response is conserved in primary murine macrophages, in which mimics of physiological stress and classical activation stimuli also induce Blimp-1. During the UPR, BLIMP-1 mRNA is induced at the level of transcription, with enhanced recruitment of RNA polymerase II to the BLIMP-1 promoter. Furthermore the stress response is limited to induction of BLIMP-1α mRNA and does not affect levels of an alternate transcript encoding a truncated protein, BLIMP-1β. The common induction of BLIMP-1 mRNA by stimuli which trigger the UPR supports the hypothesis that BLIMP-1 is a target of the eIF2α kinase family. To test this hypothesis directly, we employed a dominant negative mutant PERK. Our data demonstrate that the BLIMP-1 response to UPR stress is dependent on an intact PERK signaling pathway. Collectively our results provide evidence for a novel link between cellular stress, the eIF2α kinase family and a regulator of differentiation in macrophages and B-cells.

Description: Background: Myelodysplastic syndromes (MDS) are associated with cytogenetic clones. To follow the maturation sequence of original clones and evolved subclones with additional cytogenetic abnormalities, progenitor cells, immature and mature myeloid cells were sorted by flow cytometry and analyzed separately by fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH). Methods: Flow cytometry sorted cell fractions from bone marrows for sixteen patients with MDS-associated cytogenetic abnormalities were evaluated by FISH. Flow cytometric cell sorting was based on CD34+/low side scatter (SS) for progenitors, CD13+/CD16-/high SS for immature myeloid cells, and CD13+/CD16+/high SS for mature myeloid cells. Customized color labeling (spectrum orange, green and aqua) of FISH probe combinations were designed to detect and to analyze clonal evolution for each patient based on their known cytogenetic abnormalities and clonal evolution patterns. Three marrow aspirates were sorted for analysis by SNP/CGH microarray. Results: The 16 bone marrow specimens evaluated by FISH were categorized into three groups: (1) eight patients with a single, good-to-intermediate cytogenetic abnormality; (2) four patients with monosomy 7; and (3) four patients with more than one chromosome abnormality with evidence of clonal evolution by conventional cytogenetic analysis, excluding monosomy 7. The Group-1 abnormalities included deletion 20q (n=4), trisomy 8 (n=2), deletion 5q (n=1), and trisomy 11 (n=1). All specimens from this group showed FISH abnormalities in equal proportions in myeloid progenitors, immature and mature myeloid cells. Group-2 had four patients with monosomy 7. All four had monosomy 7 concentrated in the progenitor cells (45-79%) compared to immature and mature myeloid compartments (less than 9-36%). For Group-3, known original clones with single cytogenetic abnormalities (deletion 20q, monosomy 3, deletion 7q or 5q) were monitored by FISH analysis. Using customized FISH panels, the presence of subclones with additional cytogenetic aberrations (trisomy 8 in three patients and gain of a marker chromosome characterized by the centromere of chromosome 4 in a fourth patient) was assessed using single-cell resolution. The progenitor and immature myeloid compartments had the original founding clones containing a single cytogenetic abnormality at 15-34% and the subclones with the additional aberrations at 23-76%. In contrast, the mature myeloid cells were comprised of the original clone at 20-40%, but the subclones with additional aberrations were absent in the mature myeloid compartment in three patients and reduced by more than half in a fourth. For three patients sorted bone-marrow fractions were analyzed by SNP/CGH microarray. In one patient with trisomy 8 as the sole abnormality, the same aberration was observed in both the immature and mature myeloid compartments. In two other patients, additional abnormalities not seen in the mature myeloid cells were detected in the progenitor and immature myeloid compartments. Conclusions: These data show two main patterns for the distribution of clonal cytogenetic abnormalities among myeloid cells in MDS: 1) A continuous distribution at all stages of maturation was found for single aberrations with good-to-intermediate prognostic associations (Group 1) and 2) A disrupted distribution pattern with accumulation of the cytogenetic aberrations in the progenitor compartment as compared to the immature and mature myeloid compartments for specimens characterized by monosomy 7 (Group 2). Similarly, subclones characterized by additional cytogenetic abnormalities (Group 3) were sequestered in the progenitor and immature myeloid compartments while the original founding clone was evenly distributed throughout maturation. These data demonstrate that specific cytogenetic abnormalities associated with poor prognosis (e.g. monosomy 7) as well as acquired cytogenetic abnormalities as a result of clonal evolution can cause disruption of myeloid cell maturation in MDS. Disclosures Zehentner: HematoLogics Inc.: Employment, Equity Ownership. Hartmann:Hematologics Inc.: Employment. Johnson:Hematologics Inc.: Employment. Bennington:HematoLogics Inc.: Employment. Fritschle:HematoLogics Inc.: Employment. Ghirardelli:HematoLogics Inc.: Employment. Broderson:HematoLogics Inc.: Employment. Chapman:Hematologics Inc.: Employment. Stephenson:Hematologics Inc.: Employment. de Baca:Hematologics Inc.: Employment. Singleton:Hematologics Inc.: Employment. Wells:HematoLogics Inc.: Employment, Equity Ownership. Loken:Hematologics: Employment, Equity Ownership.

Description: Activation of haemostasis in sepsis may lead to microvascular thrombosis and progression to multiorgan failure (MOF). Almost all critically ill patients with sepsis have abnormal coagulation screens but these are unlikely to adequately represent the state of a patient’s haemostatic system and global assays may be more useful. Normal controls (n=32) and critically ill patients with sepsis (n=39) were recruited. Coagulation factors, antithrombin (AT) and protein C (PC) were measured. Thrombin generation was measured using the calibrated automated thrombogram (CAT) in platelet rich (PRP) and poor (PPP) plasma. Low dose tissue factor (6pM) activated whole blood Rotem® was measured and the first derivative of the trace gave a velocity of clot firmness. Haemostatic changes in sepsis compared to normal controls Controls Controls mean (SD) Sepsis mean (SD) P Apparent effect of change PT (s) 11.7 (0.5) 19.7 (5.9) 0.001 Anticoagulant aPTT (s) 27 (3.4) 44.1 (18.2) 0.001 Anticoagulant Fibrinogen g/l 2.8 (0.57) 5.3 (2.1) 0.001 Prothrombotic FII IU/dl 100 (12.1) 64 (32.2) 0.001 Anticoagulant FV IU/dl 116 (22.9) 96 (55.2) 0.03 Anticoagulant FVII IU/dl 130 (31.1) 58 (33.5) 0.001 Anticoagulant FVIII IU/dl 107 (31.5) 242 (96) 0.001 Prothrombotic FIX IU/dl 101 (16.5) 112 (51.3) NS Neutral FX IU/dl 123 (16.6) 75 (42.1) 0.001 Anticoagulant FXI IU/dl 116 (15.7) 80 (41) 0.001 Anticoagulant FXII IU/dl 125 (27.8) 56 (29.4) 0.001 Neutral PC % 127 (20) 66 (37) 0.001 Prothrombotic AT IU/dl 103 (8) 64 (29) 0.001 Prothrombotic CAT in PRP Lag time (min) 17 (8) 30 (23) 0.02 Delayed ETP (nM.min) 1395 (488) 1270 (573) NS Neutral Peak thrombin (nM) 76 (40) 55 (31) 0.02 Anticoagulant Time to peak (min) 32 (12) 50 (29) 0.001 Delayed CAT in PPP Lag time (min) 2.4 (0.9) 5.1 (5.4) 0.001 Delayed ETP (nM.min) 1681 (281) 1645 (442) NS Neutral Peak thrombin (nM) 454 (100) 343 (146) 0.001 Anticoagulant Time to peak (min) 4.2 (1.2) 6.8 (6.6) 0.001 Delayed Low dose tissue factor Rotem Clot time (s) 818 (271) 1170 (766) 0.04 Delayed Alpha angle (°) 51 (12) 67 (17) 0.005 Prothrombotic MCF (mm) 51 (12) 67 (17) 0.001 Prothrombotic Max vel (mm/s) 6.5 (3.0) 10.9 (7.4) 0.005 Prothrombotic Time to Vmax (s) 1040 (334) 1079 (650) NS Neutral AUC 51 (12) 62 (24) 0.001 Prothrombotic The results show that despite decreased levels of factors II, V, VII, XI and XII (correlation with decreased albumin, P

Description: Oral mucositis (OM) is a common complication in cancer patients receiving chemotherapy (CT). OM is characterized by damage to the epithelium of the oral-pharyngeal cavity. CG53135-05, a recombinant human fibroblastic growth factor 20 (rhFGF-20) protein, has demonstrated epithelial and mesenchymal cell proliferation stimulating activity in vitro and reduces OM using single doses in a hamster mucositis model. The goal of this dose-escalating tolerance study was to assess the safety, tolerability and pharmacokinetics of CG53135-05 in cohorts of four patients (pts) at 0.03, 0.1, 0.33, and 1mg/kg (concentrations are determined by the UV method which are equivalent to 0.1, 0.3, 1.0, and 3.0 mg/kg by the Bradford method previously used). Dose escalation was stopped due to tolerability information at 0.33 mg/kg delivered in 15 min (reported in another Phase I study) and the protocol was amended to add a 0.2 mg/kg dose. The World Health Organization (WHO) OM and OMAS scoring systems were used to measure the incidence and severity of OM. The interim results are reported: Ten pts received CG53135-05 at 0.03 mg/kg (n=4) or 0.1 mg/kg (n=6) as a single 100-ml intravenous infusion administered 3 days after completion of the CT. After infusion, CG53135-05 reached maximum plasma concentration within 1h. The mean terminal exponential half-life was 49 min (range: 16.2–87 min, n=5). No pts discontinued the trial due to adverse experiences. Adverse events (number pts) that may be related to the study drug include: nausea (2); chills (2); fever (2); vomiting (1); dizziness (1); vision - “lights flashing” (1) and astigmatism (1); neuropathy (1); tachycardia (1); headache (1); and premature atrial complex (1). All reported incidences were mild to moderate. No grade 3 or 4 laboratory toxicity associated with the study drug was noted. Three serious adverse events deemed unrelated to study drug were noted from two pts including cancer progression, catheter infection, and small intestinal obstruction. Among the treated pts, no grade 3 or 4 OM was observed. Three pts developed Grade 2 mucositis that lasted between 1–5 days. No pts required total parenteral nutrition. Approximately 72–84% of pts receiving CPT-11 experience diarrhea. Of the six pts who received CPT-11 as part of CT in this trial, two pts (both receiving 0.03mg/kg of CG53135-05) experienced mild to moderate diarrhea. Conclusions: CG53135-05 is well tolerated with single dose administration at 0.03 and 0.1 mg/kg. Dosing at 0.2 mg/kg is ongoing. The full results of this study will be presented.