Academic literature on the topic '060199 Biochemistry and Cell Biology not elsewhere classified'

Abstract Background Patients with solid tumors treated at a National Cancer Institute designated cancer center (NCI-CC) have been found to have improved survival when compared to those treated elsewhere. Few population-based studies have evaluated the association between location of care, complications associated with intensive therapy and early mortality (defined here as death ² 60 days from diagnosis) in patients with AML. Methods Using linked data from the California Cancer Registry and Patient Discharge Dataset from 1999 to 2012, we identified patients ³15 years of age diagnosed with AML who received inpatient treatment. Hospitals where patients received their care were classified as either NCI-CC or non-NCI designated facilities (non-NCI-CC). Logistic regression was used to estimate associations between patient characteristics (age, sex, race/ethnicity, year of diagnosis, marital status, neighborhood socioeconomic status, health insurance, and medical comorbidities) and location of care (NCI-CC vs non-NCI-CC facilities), and then to build a propensity score. Inverse probability weighted logistic regression models were used to determine associations between location of care and complications with early mortality. Interactions of complications and location of care with early mortality were also considered. Results are presented as adjusted odds ratios (OR) and 95% confidence intervals (CI). Results Of the 5613 patients with AML identified, 1406 (25%) were treated at an NCI-CC. Compared to patients treated elsewhere, AML patients treated at an NCI-CC were more likely to be <65 years of age, Hispanic (versus non-Hispanic white), live in higher socioeconomic status neighborhoods, have fewer comorbidities and have Medicare or public (versus private) health insurance. Patients treated at NCI-CCs had higher rates of renal failure (21% vs 18%, P=0.03) and lower rates of respiratory failure (11% vs 14%, P=0.002) and sepsis (33% vs 36%, P=0.04) than those treated at non-NCI-CCs. Rates of bleeding, thrombosis, liver failure and cardiac arrest did not differ by location of care. After propensity-score weighting, baseline characteristics were balanced between patients treated at NCI-CCs versus non-NCI-CCs (all standardized mean differences <2.3%). In multivariable, inverse probability weighted models, treatment at an NCI-CC (versus non-NCI-CC) was associated with lower early mortality (OR 0.50, CI 0.42-0.59) (Table). Complications associated with higher early mortality, regardless of treatment location, included: bleeding (OR 1.91, CI 1.54-2.37), liver (OR 3.41, CI 1.91-6.09), renal (OR 2.39, CI 1.97-2.90) and respiratory (OR 6.08, CI 4.88-7.57) failure and cardiac arrest (OR 15.28, CI 8.11-27.78) (Table). The impact of complications on early mortality did not differ by location of care with the exception of respiratory failure (P for interaction=0.001); AML patients with respiratory failure had a higher odds of early mortality when treated at non-NCI-CCs (OR 8.59, CI 6.66-11.07) versus NCI-CCs (OR 4.43, CI 2.68-7.33). Other factors significantly associated with early mortality included younger age, treatment after 2002, non-Hispanic White race/ethnicity (versus African Americans, Hispanics and Asians), single marital status (versus married), residence in low socioeconomic status neighborhoods (versus high) and presence of any comorbidities (versus none). Conclusions In patients with AML, initial treatment at a NCI-CC is associated with 50% reduction in early mortality than treatment at a non-NCI-CC, adjusted for baseline socio-demographic variables, comorbidities and complications within 60 days. Lower early mortality may result from differences in supportive care practices, hospital or provider experience and access to clinical trials at NCI-CCs. Future studies should evaluate the specific differences in care at NCI-CCs to inform strategies to improve early mortality outcomes for all AML patients. Table. Table. Disclosures No relevant conflicts of interest to declare.

Abstract Introduction Multiple Myeloma (MM) is considered a malignancy of post germinal center long-lived plasma cells. Nevertheless T-cell independent antigen stimulation before the exposure of the B-cell to the germinal center can happen and results to IgM secreting short lived plasma cells and lymphoplasmacytes representing thus a potential alternative normal counterpart for IgM plasma cell dyscrasias. IgM myeloma is an infrequent subtytpe of MM with an estimated prevalence of 0.5%. Due to its rarity little is known about its characteristics and prognosis in comparison with Waldestrom’s macroglobulinemia (WM) and the other MM subtypes. Purpose To identify the characteristics and the prognosis of IgM MM, and compare it predominantly with WM and subsequently with the rest of the MM subtypes. Methods We interogatted our Multiple Myeloma Data Base for cases of IgM MM and their respective Overall Survival (OS), Progression Free Survival (PFS), bone disease as defined by x-Rays, PET-CT and MRI, Gene Expression Profile (GEP), and common disease characteristics (anemia,calcium, creatinine) and compare it to the prognosis of WM and non-IgM MM. Diagnosis was based on the morphological and immunophenotypical findings of pathologically examined biopsy specimens along with the presence or not of typical clinical characteristics of MM (lytic bone lesions, hypercalcemia, renal failure) or typical clinical characteristics of WM (organomegaly, lymphadenopathy). Results There were 22 confirmed IgM MM cases. 14 of them presented at MIRT at initial diagnosis while 8 had previously been treated elsewhere. Osteolytic bone lesions and/or pathological fractures by x-ray and CT examination were evident in 16 cases. For the remaining 6 cases active bone focal lesions by either MRI or PET were identified in three. There was no organomegaly evident in cases with an available PET/CT at baseline, while only one had evidence of hilar and mediastinal lymphadenopathy along with calcified lung nodules. Elevated creatinine levels (>2.0 mg/dl) were evident in 4 cases at initial diagnosis. Their disease characteristics are depicted in the table 1. Median OS for IgM MM was 4.9 years while PFS could not be accurately estimated due to lack of data on patients treated elsewhere. Median OS for a historical control of 158 WM cases in MIRT was 9.2 years (Clin Lymphoma Myeloma Leuk. 11(1):139-42). Median OS of the WM group remained largely unaffected, even when the subgroup of the WM cases requiring treatment was analyzed (9.0 years).To further clarify if the IgM MM differs in terms of OS from the other isotypes of MM, we compared the IgM group to a group of 61 non-IgM MM cases which were matched by important prognostic clinical factors (age, creatinine> 2mg/dl, LDH>190u/L, b-2M >5.5mg/dl and Albumin<3.5gr/dl). No statistical difference was found for OS (p=0.846). Out of 22 cases, 14 of them had available GEP data on initial diagnosis. In 6 of these cases the cyclin D1 gene expression was high enough to be consistent with a t(11;14) translocation at FISH analysis, one case was consistent with a t(14;16) translocation, one with a t(4;14) translocation and two more were classified as belonging to the hyperdiploid subgroup. A comparative genomic analysis was performed on the IgM MM, the non-IgM MM and WM cases with available GEP data at initial diagnosis (14, 61 and 42 cases respectively). 1155 probesets that had expression level significantly different between WM and non IgM MM (FDR<3E-06) were identified. Then, the expression values of these 1155 probesets in all GEP samples, including WM, non IgM MM, and IgM MM, were used to build a clustering tree. We found that IgM MM mainly clustered with non IgM MM, supporting the findings of the clinical data. Conclusion IgM MM is a discrete clinical entity that should be distinguished from WM. Bone disease is evident in the majority of the cases, especially when specialized radiological techniques are incorporated at the initial work up. It holds a distinct prognosis from WM, while when balanced for prognostic factors that hold importance in MM it does not differ from the other MM isotypes. Finally analysis of the genetic data further supports the resemblance between IgM MM and the non IgM MM, and the difference with WM. Disclosures: Zhang: University of Arkansas for Medical Sciences: Co-inventor of the DNA probes for FISH of IGHC/IGHV (14q32), MMSET/FGFR3 (4p16), CCND3 (6p21), CCND1 (11q13), MAF (16q23), and MAFB (20q12) loci, sub. to the US Patent & Trademark Office as Prov. App# 61/726,327: Methods of Detecting 14q32 Translocations, Co-inventor of the DNA probes for FISH of IGHC/IGHV (14q32), MMSET/FGFR3 (4p16), CCND3 (6p21), CCND1 (11q13), MAF (16q23), and MAFB (20q12) loci, sub. to the US Patent & Trademark Office as Prov. App# 61/726,327: Methods of Detecting 14q32 Translocations Patents & Royalties.

Abstract Background: Early interim-PET (PET-2) is the most powerful factor able to predict treatment outcome in advanced-stage Hodgkin Lymphoma (HL) patients treated with ABVD (doxorubicin, bleomycin, vinblastine, and dacarbazine). The 2-y PFS of PET-2 positive patients is only 12%, but the optimal treatment for this patient subset is still unknown. For this reason in January 2006 a treatment policy was designed by GITIL (Gruppo Italiano Terapie Innovative nei Linfomi) to early intensify chemotherapy with BEACOPP (bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine, and prednisone) for HL patients with a PET-2 positive after 2 ABVD cycles. Patients and methods: 136 HL patients with advanced-stage or intermediate-stage with adverse prognostic factors (more than 3 nodal sites, ESR &gt; 50 mm, sub diaphragmatic presentation, bulky lesion), consecutively admitted to nine GITIL Italian centers were treated with two ABVD courses and re-evaluated with interim-PET. Twenty-one of these 136 patients proved to be PET-2 positive, and 19/21 are the object of this analysis. The mean age was 31.7 years (16–64), 10 patients were in stage II, 9 in stage III–IV. Bulky disease was present in 11, and B-symptoms in 16. Fourteen patients showed an IPS score 0–2, 5 patients a score 3–7. The median interval between the end of second ABVD course and PET-2 was 11 days (5–14). BEACOPP (4 escalated and 4 standard cycles) was followed by consolidation radiotherapy in 3 patients for bulky mediastinal tumor. PET scan were centrally reviewed by two well-experienced nuclear medicine experts, using the blood pool mediastinal structures (BPMS) as reference for the residual uptake, as elsewhere published (Gallamini, JCO 2007). Results. Upon central review, 2 interim-PET scan were classified as minimally positive in 2 patients, with a single MRU (Minimal Residual Uptake) lesion showing a SUV (Standardized Uptake Value) lower than BPMS, and therefore only 19 cases were suitable for the BEACOPP salvage treatment. After a mean follow-up of 14.3 months (7.0–30.2), 15 patients remain in continuous CR and 4 had a treatment failure because of disease relapse (1) or progression (3). For the responding patients the mean duration of CR was 13.0 months (6.5–30.2). The IPS score for progressing patients was 2, 3, and 4; for the relapsing-one, 0. At time of this analysis, l8 patients are still alive and 1 died during BEACOPP treatment for disease progression. The 1-year second treatment failure-free survival (1-y 2TFFS) was 94.7 % (95% C.I. 84.7–100). The 1 y OS survival was 93.3 (95%C.I. 80.7–100). In univariate analysis the only clinical factor related to treatment failure was the presence of extra-nodal disease. (Log-rank=6.8 p=0.009). Conclusions. BEACOPP-escalated regimen was able to induce durable CR in most (15/19) HL patients with a positive interim-PET. These results, although requiring a longer follow-up and a higher number of patients, seem to suggest that the very-poor prognosis of PET-2 positive, ABVD-treated HL patients can be substantially improved by early chemotherapy intensification; escalated-BEACOPP is likely to represent a good treatment choice for this very poor prognosis patient subset.

Abstract Introduction: ELANE neutropenia represents the cause of 25-30% of the cases of congenital neutropenia. Classically, its appears in the literature as in OMIM, under two distinct entities: Severe congenital neutropenia (SCN) and cyclic neutropenia (CyN). The delineation between the 2 entities is the "cyclicity" i.e. the periodic variation of the absolute neutrophils count (ANC), also called a 21-day time clock 1. However, it is extremely difficult to obtain enough sequential complete blood counts (CBC) at the onset of the disease, during an enough length period, while the patient is not experiencing a severe infection or receiving GCSF therapy. The purpose of our study is to analyze the ANC periodicity at the onset of the disease, prior to the initiation of GCSF in a cohort of patients with ELANE neutropenia. Methods : Available data from the patients, with ELANE class 4 and 5 variants, enrolled in the French Severe Chronic Neutropenia Registry, were analysed. The final diagnostic of CyN and SCN was performed considering all the follow up period (median 16.7 years) and based on the presence of recurrent periodic variation of ANC in the absence of GCSF therapy. CyN was defined as multiple documented ANCs &gt;500 cells/mm 3 , with intermittent ANC variation (n=49), while SCN is defined as ANC persistently &lt;500 cells/mm 3 (n=94). In case of irregularity (i.e. not a regular periodic pattern during all the follow up), the classification takes in consideration the majority of the follow up. A comprehensive analysis of the infectious profile is available elsewhere 2. We were focused here on the diagnostic period (roughly the 2 first months since the diagnosis). We have analysed the initial blood count of the patients and cast the patients by categories if at least 4 ANCs can be evaluated. ANC oscillations defined 4 groups: Group 1: oscillation of ANC values above and below 500 ANC/mm 3 for at least 2 cycles lead to consider the patient as Cyclic; Group 2: clear oscillation of ANC values above and below 200 ANC/mm 3 (but ever&lt;500 ANC/mm 3) for at least 2 cycles; Group 3: no oscillation of ANC values whose level are ever below 500 ANC/mm 3; Group 4: Early GCSF treatment. Results : Among the 143 patients enrolled in this study (Table 1), 137 have at least 4 CBC evaluable during the diagnosis period, including 30 who have been treated almost front line after diagnosis of neutropenia by GCSF hampering evaluation of periodicity. Such patients were all initially considered as SCN. Among the 67 finally classified as SCN, 28 (27.38%) showed an oscillation pattern (group 1), 14 (15.48%) showed minor oscillations (group 2), while 25 (30.95%) had a persistent and severe neutropenia (group 3). Among the 40 CyN, 1 have showed minor oscillations (group 2) below 500/mm 3, while 3 had a persistent and severe neutropenia (group 3). Globally, 32 /107 patients were miss- classified at diagnosis compare to the final diagnosis. Additional data shows that many health indicators could not be deducted from the initial classification like the infections rate, the use of GCSF, the death rate, the sequels rate. Conclusions: Periodic variation of ANC despite being the criteria to define the sub type of ELANE neutropenia is difficult to evaluate at the initial presentation of the disease. In addition, cyclicity is not a permanent feature in ELANE neutropenia, some patients being cyclic only for a certain time in their life span. It results a high rate of miss classification if we compare the initial diagnostic period and all the medical history of the patients. Noteworthy, the rate of several severe complications is not so clearly different between diagnosis sub categories. We propose to consider ELANE neutropenia as a unique disease characterized by a clinical spectrum ranging from more severe forms (corresponding to SCN) to milder forms, the latter often characterized at onset by ANC fluctuations. In addition, some intermediate severity forms could be characterized by minor oscillations. References 1 Horwitz M, Benson KF, Person RE, Aprikyan AG, Dale DC. Mutations in ELA2, encoding neutrophil elastase, define a 21-day biological clock in cyclic haematopoiesis. Nat.Genet. 1999;23:433-436. 2 Rotulo GA, Plat G, Beaupain B et al. Recurrent bacterial infections, but not fungal infections, characterise patients with ELANE-related neutropenia: a French Severe Chronic Neutropenia Registry study. Br J Haematol. 2021 doi: 10.1111/bjh.17695 Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Abstract Introduction: The revised cytogenetic risk stratification of patients with primary myelofibrosis (PMF) divided patients into 3 prognostic categories, with additional new category of very high risk patients (VHR). This score should enhance traditional classification incorporated in the Dynamic International Prognostic Scoring System-Plus (DIPPS-Plus). Objective: To evaluate the prognostic utility of cytogenetic stratifications (DIPSS-Plus and the revised cytogenetic model) in patients with PMF referred to our institution between 1984 and 2016. Methods: We retrospectively reviewed the charts of 883 patients with PMF with available cytogenetic analysis at the time of referral to our institution (> 10 metaphases). Cytogenetic was reported according to the International System for Human Cytogenetic Nomenclature. Patients were classified into cytogenetic risks based on DIPSS-Plus (Gangat, JCO, 2011), and the revised cytogenetic model (Tefferi, Leukemia, 2017). Overall survival (OS) was estimated using the Kaplan-Meier method, and groups were compared by the log rank test. Impact of cytogenetic abnormalities on OS was also evaluated by comparing them against patients with diploid karyotype using stepwise Cox regression. Results: Median age was 66 years (range, 27-88), and 64% of patients were male. The distribution of DIPSS scores was as follows: 8% low, 48% intermediate 1, 44% intermediate 2 and 14% high. OS in each DIPSS category was 53, 46, 26, 15 months (p<0.001). The JAK2, MPL and CALR mutation was present in 55% (n=486), 6% (n=50), and 7% (n=64). Overall, 563 (64%) patients had diploid karyotype. The most frequent abnormal karyotypes were single 20q- (n=68, 8%), single 13q- (n=40, 4.5%), and ≥3 abnormalities (Abn; complex karyotype, CK, n=52, 6%). Among patients with CK, 27 (52%) pts had VHR Abn. After a median follow-up of 22.4 months (range, 0.5-251); 708 (80%) of patients died. Eighty five patients (10%) developed acute leukemia, 39% of these patients had CK. According to DIPSS-plus, patients were stratified into favorable (FAV, n=758, 86%) and unfavorable (UNF, n=126, 14%) category with distinct median OS of 35 months (range, 31-39), and 17 months (range, 11.6-22), p < 0.001 (HR 1.37, [95% CI 1.11-1.7]). Three year OS was 49% and 32%, respectively (Figure 1a). The revised cytogenetic stratification classified patients into favorable (n=687, 78%), unfavorable (n=151, 17%), and VHR (n=47, 5%) with respective OS of 35, 32 and 10 months (overall p<0.001, FAV vs UNF p= 0.8; Figure 1b); similar between patients in favorable and unfavorable groups. Three year OS for each group was 49%, 46% and 12%, respectively. OS of patients with individual cytogenetics (as used in the revised classification) is depicted in Table. Patients with single deletion 13q have significantly inferior OS than the remaining patients in FAV group. Patients with sole abnormality of chromosome 1 and trisomy 9 had the longest OS within the FAV group, but without reaching a statistical significance. Similarly, patients with sole trisomy 8, sole deletion 7q/5q, and other sole Abn not included elsewhere, had inferior OS when compared to the remaining patients in UNF group (Table 1). After re-grouping patients with different OS from FAV and UNF groups, we have noticed an intermediate group of patients containing the above mentioned Abn with distinctly different OS from FAV and UNF group of 24 months (range, 14.5-33; Figure 1c). Conclusions: Results from our cohort of 883 PMF patients did not confirm better discriminatory power of revised cytogenetic stratification model when compared to the DIPSS-Plus, as it failed to differentiate different OS between favorable and unfavorable groups. In our cohort, patients with single deletion 13q, single trisomy 8, and abnormalities of 5q/7q have superior OS to very high risk patients, but inferior to all remaining patients. Because the revised cytogenetic stratification has been already incorporated into newer complex molecular prognostic models of patients with PMF (MIPSSversion2.0, GIPSS), its further validation is warranted. Table Abbr.: Chr, chromosome, del, deletion, DUP, duplication, transl, translocation, excl, excluding; ¥OTHER solo: INV(9) in [3], Abn chr. 11, 12, 16, 17, 18 (mostly deletion of p/q arms, or addition) [7]; VHR = very high risk (-7; inv(3)/3q21; i(17q); 12p-/12p11.2; 11q-/11q23; autosomal trisomies excl. +8/+9). Disclosures Bose: Incyte Corporation: Honoraria, Research Funding; CTI BioPharma: Research Funding; Astellas Pharmaceuticals: Research Funding; Constellation Pharmaceuticals: Research Funding; Pfizer, Inc.: Research Funding; Celgene Corporation: Honoraria, Research Funding; Blueprint Medicines Corporation: Research Funding. Pemmaraju:plexxikon: Research Funding; cellectis: Research Funding; Affymetrix: Research Funding; daiichi sankyo: Research Funding; stemline: Consultancy, Honoraria, Research Funding; novartis: Research Funding; samus: Research Funding; celgene: Consultancy, Honoraria; abbvie: Research Funding; SagerStrong Foundation: Research Funding. Cortes:novartis: Research Funding. Verstovsek:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy; Italfarmaco: Membership on an entity's Board of Directors or advisory committees.

Abstract Background: Adult T-cell leukemia-lymphoma (ATL), which is a hematological malignancy related to human T-lymphotropic virus type I (HTLV-1), is known as a malignant lymphoid disease with poor prognosis. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is considered a treatment modality to contribute prolonging the survival in some population of patients (Katsuya H et al. 2015 Blood). A small study indicated that ATL cells frequently have chromosomal abnormalities including various numerical aberrations and complex structural abnormalities (Kamada N et al. Cancer Res. 1992). However, there has been no large study to examine the relationship between chromosomal abnormalities and survival. Here we report the impact of chromosomal abnormalities on survival in ATL patients with conventional chemotherapy. Patients and Methods: In this study, we extracted a population of patients from the database of ATL-PI project, which is a nation-wide survey of ATL patients newly diagnosed between January 2000 and May 2009, and the overall results were reported elsewhere (Katsuya H et al. JCO 2012, Blood 2015). Fifteen hundred and twelve patients were registered in this project, and we excluded the patients who received allo-HSCT, and who were diagnosed with smoldering and chronic type ATL. Moreover, patients whose chromosomal analysis with G-banding stain showed normal, multiploid or no mitosis, or no information on chromosomal analysis and on the factors to determine the ATL-PI were also excluded. As a consequence, 210 patients with acute (n=157) and lymphoma (n=53) types were selected for this analysis. Abnormal karyotypes were analyzed from the aspect of numerical and structural break points. To identify the specific abnormalities and to avoid the secondary changes, the stem-line karyotype, which meant the simplest abnormal clone, was used as the representative karyotype for this study. A statistical analysis was performed with 'EZR' (Kanda T. BMT 2013). We examined an overall survival (OS) in relation to chromosomal abnormalities using cox proportional hazard regression model, and p-value of less than 0.05 was considered as statistically significant. Results: Among the 210 patients, 119 and 91 patients were male and female, respectively. The median age of patients was 66 years (40-89). With respect to other factors of ATL-PI, the median level of soluble interleukin-2 receptor (sIL-2R), serum albumin, were 30800 U/mL (620-1,219,000), 3.6 g/dL (1.6-5.3), respectively. Almost all patients were diagnosed with stage II-IV of clinical stage (n=200), and almost half of patients certified 2-4 of ECOG-PS (n=113). Sixty-nine, 106, and 35 patients were classified as high, intermediate and low risk by ATL-PI, respectively. Specimens used for chromosomal analysis were taken from lymph nodes (n=77), bone marrow (n=72), peripheral blood (n=50), pleural fluids (n=4), ascites (n=4) and 1 each of stomach, sinusoidal tumor, and tonsillar lesions. The median number of numerical abnormalities and marker chromosomes were 2 (0-20) and 1 (0-20), and the median number of structural break points was 6 (0-21). The numerical abnormalities were loss of sex chromosomes (31%) followed in decreasing order by -14 (24%), -13 (19%), -9 (12%) and -17 (12%) while the structural break points were located at 1p (30%), 3q (30%), 14q (30%), 6q (26%) and 1q (22%). The median OS was 286 days (1-2565). ECOG-PS, serum albumin level, sIL-2R, and structural break points at 9q or 19p, and the number of structural break points over 4 or 5 points were shown to be the significant factors affecting the survival by univariate analysis. Then, the multivariate analysis indicated that 2-4 of ECOG-PS (HR: 2.021, 95%CI: 1.436-2.791, p<0.001), serum albumin level < 3.5 g/dl (HR: 1.631, 95%CI: 1.282-2.074, P<0.001), and chromosomally structural break points at 9q (HR: 1.584, 95%CI: 1.096-2.289, p<0.001) and 19p (HR: 1.765, 95%CI: 1.038-2.999, p<0.001) were factors that negatively contributed to OS. Discussions and Conclusion: This is the first large study showing the impact of chromosomal abnormality in acute and lymphoma types ATL on OS. It demonstrated that structural break points at 9q and 19p were the independent risk factors for OS in addition to those of ATL-PI. Disclosures Katsuya: The Uehara Memorial Foundation: Research Funding. Utsunomiya:Daiichi Sankyo Co., Ltd.: Speakers Bureau. Tsukasaki:Daiichi Sankyo Co., Ltd.: Consultancy; Takeda: Research Funding. Suzumiya:Takeda: Honoraria; Astellas: Research Funding; Kyowa Hakko kirin: Research Funding; Chugai: Honoraria, Research Funding; Toyama Chemical: Research Funding; Eisai: Honoraria, Research Funding.

Abstract Ph chromosome is the hallmark of CML. However there are few reports of additional chromosomal abnormalities at the time of diagnosis and the impact this has on overall survival (OS). [NT1] The Cytogenetic laboratory at this hospital provides a regional service as a single facility. The data from this laboratory was combined with survival information to evaluate the impact of additional chromosomal changes on outcomes in patients with CML. Methods: This is a retrospective population based study, it was not possible to obtain consent from individual patients and details about haematological parameters or treatments delivered were not available. The aim was to evaluate if cytogenetic changes should be considered in addition to established risk scoring systems. Patients were classified as complex-Philadelphia (Ph) if they had t(9;22)(q31;q34) with additional chromosomal abnormalities. Impact of individual additional abnormalities was analysed and then the effect was stratified according to presence of chromosomal gains, deletions or translocations. Few cases who had normal cytogenetics on their first sample as they were initially treated elsewhere. Results: 1129 patients were diagnosed with CML between 1985 and 2013, with 4760 samples analysed. The median age of the patient was 52.4 years (4.3-103, 602 male; 511 female; 16 unknown). Median follow up was 6.4 years [0-26.8 years, 725/1129 (64.2%) had follow-up more than 10 years]. End point for analysis was probability of survival at 10yr. 194/1129 (17.2%) had complex-Ph at diagnosis, 759/1129 (67.2%) had standard Ph, 77/1129 (6.8%) had negative cytogenetics and in 34/1129 (3%) cytogenetic analysis failed at diagnosis. Patients with standard Ph translocation had significantly better chance of achieving cytogenetic CR than those with complex-Ph (23.4% vs. 13.4%, p<0.001). OS was significantly better in patients below the age of 45 (65% vs 25% p <0.0001). OS was also better in patients diagnosed after 2000 (67 % vs 40 %, p<0.0001). In univariate analysis OS was significantly lower with trisomy 8 (10% vs 50%, p<0.0001), del(5q) [NT2] (20% vs 50%, p=0.001), other deletions (12% vs 48%, p=0.0005), del 17( 48% vs. 0%, p<0.0001), add 21 (50% vs. 0%, p<0.0001), any translocations (50% vs. 22%, p<0.0001), der 22 or iso 17 (48% vs. 10%, p<0.0001), any deletions (50% vs. 20%, p<0.0001) and variant Ph translocations (50% vs 22%, p=0.004). In multivariate analysis, excluding year of diagnosis, age group (HR 1.93, 95% CI:1.6-2.4 P=<0.0001), complex t(9;22;v) (HR 1.8, 95% CI:1.0-3.1, P=0.035), del(17q) (HR 3.8, 95% CI:1.1-12.6, P=0.033), translocations (HR:1.6, 95% CI: 1.1-2.25, p=0.013), trisomy 8 (HR: 1.76, 95% CI: 1.19-2.65, p=0.005), add 21 (HR: 3.21, 95% CI: 1.65-6.25, p=0.001) and der 22 or iso17 (HR: 1.57, 95% CI: 1.1-2.25, p=0.013) were independently associated with inferior OS. Number of risk factors in individual patients was used to design a scoring system. Patients with 0 risk factor (70% OS at 10 yr.), 1 risk factor (40% OS at 10 years), 2 risk factors (22% OS at 10 years) or more than 3 risk factors (12% OS at 10 years) had incrementally reduced OS. This was true of patients diagnosed before and after 2000. This analysis suggests that incorporating nature of karyotype at diagnosis can refine established scoring systems. However this data needs to be analysed with larger patient population to include all established risk factors and the effect of therapeutic measures. Disclosures Cavet: Novartis: Research Funding; BMS: Research Funding.

Abstract Background: 40-80 % of patients with myelodysplastic syndrome (MDS) become transfusion dependent during their disease course and are at risk for the development of alloimmunization. Red blood cell (RBC) alloantibodies can make finding compatible blood for transfusion more difficult, expensive and time consuming. Allommunization rates of approximately 30-47% have been reported in patients with sickle cell disease and the transfusion of RBCs prophylactically matched for Rh antigens E and C, and K antigens reduced the rate of alloimmunization from 3% to 0.5% per unit (Vichinsky et al, 2001). In 2007, our hospital instituted a policy of transfusing prophylactic Rh and K matched blood to MDS patients. The objectives of this study were to compare the rates of alloimmunization in MDS patients who received prophylactic Rh and K matched blood compared to those that did not and identify potential risk factors for alloimmunization. Methods: 193 Transfusion dependent MDS patients were identified out of 387 patients registered and prospectively followed in a local MDS registry. Transfusion dependence was defined as the receipt of at least 1 unit of PRBC every 8 weeks for a minimum of 16 weeks. Records of transfusions received up to May 1, 2014 were collected from blood bank databases of the hospitals at which patients were transfused. Patients were classified according to whether phenotyping had been performed, the location of transfusions (transfused only at our institution, transfused only at an outside institution or transfused at both sites) and whether prophylactic Rh (E, C antigens) and K matched blood was transfused. Data were descriptively analyzed and we conducted univariate and multivariate logistic regression using p< 0.05 as statistically significant to identify risk factors for alloimmunization. Results: 176 MDS patients with complete transfusion records are included, 73 transfused at Sunnybrook, 92 transfused in community hospitals and 11 at both. The median age was 72 yrs (range 22-89), 60% were male, and 8%, 43% and 27% had very low, low and intermediate risk R-IPSS scores respectively. Median follow up was 2.9 years (IQR 1.6-5) 3.49 SD). Blood groups O, A, B, AB and O were 45%, 38%, 15% and 2% respectively, while 85% were RhD+. The median time from diagnosis until first transfusion was 4 months (IQR: 0.2-14), with 51 patients having received at least 1 transfusion prior to diagnosis at a median time of 0.9 months. 4.5% had a pre-existing allo-antibody at time of MDS diagnosis. With a median follow up from diagnosis of 3 years (IQR:1.6-5)), the median number of RBC units transfused was 38 (IQR: 15-98)) and 36 (20%) patients developed new alloantibodies (median 2 (IQR (1-2.5) alloantibodies). The median number of RBC units until first allo antibody was 13.5 units (range 0-121) and 1.25 years from diagnosis (95% CI:0.4-2.1). The majority of the alloantibodies were in the Rh (n=28) and K (n=14) groups (80%) and co-existed 27% of the time. More patients transfused at our hospital received prophylactic Rh K matched blood sometimes or always (60% versus 26%) and rates of allo-immunization were decreased by 65% (absolute rate of alloimmunization 10% versus 29%). By multivariate analysis analysis, number of rbc transfused (p<.0001), receiving prophylactic phenotype matched blood (p=.0008) and location of transfusions (Sunnybrook versus elsewhere (p=.03)) were independent risk factors for alloimmunization. Conclusions: 20-30% of RBC transfusion dependent MDS patients will become allo-immunized to clinically significant blood group antigens, the majority being Rh and K antigens. The practice of phenotyping at baseline and prophylactically transfusing Rh and Kell matched blood decreases rates of alloimmunization up to 65% and should be strongly considered for routine transfusion practice in centres that treat MDS. Table 1. All Patients (n=176) Sunnybrook (n=73) Community(n=92) Ever phenotyped 45% 64% 28% Phenotyped before 1st transfusion 20% 38% 8% Developed allo-antibodies 20% 10% 29% Received prophylactic Rh K matched blood (developed alloantibodies) Never Sometimes Always 58% (16%) 24% (42%) 18% (6%) 40% (3%) 23% (23%) 37% (7%) 74% (22%) 22% (60%) 4% (0%) Figure 1. Allo Antibody free survival according to number of red cell units transfused in patients that developed an allo-antibody Figure 1. Allo Antibody free survival according to number of red cell units transfused in patients that developed an allo-antibody Disclosures Wells: Celgene: Honoraria, Other, Research Funding; Novartis: Honoraria, Research Funding; Alexion: Honoraria, Research Funding. Buckstein:Celgene: Research Funding.

Abstract The Bruton's Tyrosine Kinase (BTK) inhibitor ibrutinib is very effective in chronic lymphocytic leukemia (CLL). Resistance in CLL is at least in part mediated by acquired mutations in BTK or down-stream PLCG2. Here we describe the largest institutional cohort of CLL patients treated with ibrutinib, focusing on risk factors for relapse, prevalence of known resistance mutations, and development of a monitoring strategy to identify patients at risk for relapse. All 308 patients from The Ohio State University Comprehensive Cancer Center participating in 4 clinical trials of ibrutinib were included. Deep sequencing for BTK and PLCG2 was performed using Ion Torrent Personal Genome Machine and covered coding regions of both genes. Preclinical experiments used XLA cell lines (Coriell Institute) infected with lentiviral constructs containing empty vector, wild type BTK, or C481S BTK. With a median follow-up of 40.5 months (range 4-71 months), 136 patients remain on study, 14 have received transplant or therapy elsewhere, and 158 have discontinued. 83 patients experienced disease progression, classified as Richter's or PLL transformation (n=28) and progressive CLL (n=55). Using multivariable models, baseline risk factors for ibrutinib discontinuation due to transformation include complex karyotype (p<0.01) and MYC abnormalities on FISH (p=0.051). Age<65, del(17p) by FISH, and complex karyotype all associated with discontinuation due to CLL progression (p<0.05). Relapse generally has a poor prognosis, with a median survival from ibrutinib discontinuation for patients with transformation and progressive CLL of 3.9 (95% CI 2.0 to 10.1) and 22.7 (95% CI 13.5 to not reached) months, respectively. 46 patients with progressive CLL had samples at relapse available for deep sequencing. Of these, 39 had mutations in BTK and/or PLCG2 (84.8%; 95% CI 71.1-93.7). Distribution of mutation included patients with BTK C481 mutation only (n=30), mutation in PLCG2 only (n=3) and both BTK/PLCG2 genes (n=6). Given the poor prognosis at relapse and presence of acquired mutations in the majority of progressive CLL patients, we were interested to evaluate whether the emergence of small clones of mutated cells could be used as a biomarker to predict relapse. For 15 patients with BTK or PLCG2 mutations, serial samples were available prior to relapse, and a clone of resistant cells could first be detected a median of 9.3 months prior to clinical relapse (95% CI: 7.6-11.7). Based on the finding that patients who relapsed on ibrutinib often had rapid progression and poor outcomes, we initiated a clinical-grade monitoring strategy in our institutional CLIA-certified molecular lab starting in November 2014. Mutational analysis of the entire coding regions of BTK and PLCG2 was performed on a cohort of 112 patients every 3 months. To date, 8 patients have clinically relapsed and all 8 had BTK C481S mutations with expansion of the clone prior to relapse. BTK C481S mutations of over 1% allelic frequency were detected in an additional 8 patients. All patients have had expansion of the resistant clones and all but one, who discontinued therapy without clinical relapse, have had increasing circulating CLL cells in the peripheral blood. Four of these 7 patients have also had increasing lymph node size on CT scan. In 11 patients who relapsed with BTK C481S, samples were available following discontinuation. In 5 patients treated subsequently with venetoclax, allelic fractions of C481S decreased dramatically or were eliminated, but in 6 patients treated with other agents, the mutant clone persisted. To investigate whether this was due to differences in biology between wild type and C481S BTK, we created XLA cell lines without BTK protein expression stably expressing BTK, or C481S BTK. C481S BTK showed enhanced BCR signaling as evidenced by pERK and cMYC expression, and enhanced migration compared to wild type BTK (p=0.04). This demonstrates for the first time that this acquired resistance mutation fundamentally alters the tumor cell biology. These data confirm our initial findings that relapse on ibrutinib is primarily mediated through mutations in BTK and PLCG2 and suggest that the presence of these mutations may have utility as an early biomarker to predict clinical relapse. Early identification of relapsing patients could improve outcomes by facilitating initiation of adjunct therapies such as venetoclax to eliminate the small resistant clone. Disclosures Woyach: Karyopharm: Research Funding; Morphosys: Research Funding; Acerta: Research Funding. Jones:Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics, LLC, an AbbVie Company: Membership on an entity's Board of Directors or advisory committees, Research Funding. Andritsos:Hairy Cell Leukemia Foundation: Research Funding. Awan:Innate Pharma: Research Funding; Pharmacyclics: Consultancy; Novartis Oncology: Consultancy. Lozanski:Beckman Coulter: Research Funding; Stemline Therapeutics Inc.: Research Funding; Boehringer Ingelheim: Research Funding; Genentech: Research Funding.

Background Long-term PI-based treatment is associated with improved outcomes in MM. Nonetheless, prolonged therapy with parenteral PIs (e.g. bortezomib) can be challenging in the real world, with median duration of therapy (DOT) of 4-7 months. Barriers to this long-term approach may include the burden of repeated intravenous/subcutaneous administration, difficulty travelling to/accessing treatment centers (e.g. due to environmental factors, travel restrictions, social/family situations), patient preference for treatment outside of a hospital or clinic setting, comorbidities, and toxicity. The US MM-6 study (NCT03173092) is investigating in-class transition (iCT) from parenteral bortezomib-based induction to all-oral ixazomib-based therapy (ixazomib-lenalidomide-dexamethasone; IRd) in the diverse US community population with the aim of increasing PI-based treatment duration while maintaining quality of life and improving outcomes. We report updated efficacy and safety for the first 101 patients. Methods Transplant-ineligible/delayed-transplant (&gt;24 months) NDMM patients with stable disease or better after 3 cycles of bortezomib-based induction are being enrolled at US community sites (including Veterans Affairs hospitals) to receive IRd (ixazomib 4 mg, days 1, 8, 15; lenalidomide 25 mg, days 1-21; dexamethasone 40 mg, days 1, 8, 15, 22) for up to 39 x 28-day cycles or until progression/toxicity. The primary endpoint is progression-free survival (PFS); key secondary endpoints include rates of partial (PR), very good PR (VGPR), and complete response (CR), and DOT. Results As of June 1 2020, 101 patients had been treated at 21 sites. Median age was 73 years (range 48-90), with 46% aged ≥75 years; 16% and 10% were of African American and Hispanic ethnicity, respectively. Table 1 summarizes the key characteristics of these real-world patients. A total of 95% of patients had ≥1 comorbidity at the start of IRd therapy including renal and urinary disorders (38%), cardiac disorders (29%), peripheral neuropathy (PN; 14%), and diabetes mellitus (13%) (Table 2). With 53 (52%) patients remaining on therapy and enrollment ongoing, mean duration of PI therapy from the start of bortezomib-based induction was 12.4 months, and mean duration of IRd therapy after iCT was 9.2 months (Table 3). Patients have received up to 29.4 months (31 cycles) of IRd to date. The overall response rate (ORR) after bortezomib-based induction was 62% (7% CR, 32% ≥VGPR). After iCT to IRd, the ORR increased to 71%, with the CR and ≥VGPR rates increasing to 29% and 53%, respectively (Figure); of 33 patients with stable disease following bortezomib-based induction, 14 (42%) achieved CR (n=10) or VGPR (n=4) after iCT. With a median follow-up of 12 months and enrollment ongoing, 13 patients had progressed and two had died during PFS analysis. The 12-month PFS rate was 84% (95% CI, 73-91) from the start of bortezomib-based induction and 80% (95% CI, 69-88) from the start of IRd. During IRd treatment to date, 91% of patients have had treatment-emergent adverse events (TEAEs) (54% grade ≥3). Grade 3 TEAEs (≥5% of patients) were diarrhea (8%), pneumonia (7%), and syncope (5%). TEAEs led to study drug modification in 52% of patients and discontinuation in 7% of patients; 37% had serious TEAEs. Diarrhea, nausea, and vomiting occurred in 43%, 23%, and 14% of patients (8%, 2%, 2% grade 3), and led to dose modification in 11%, 5%, and 2%. PN (not elsewhere classified; high-level term) occurred in 32% of patients (2% grade 3) and led to dose modification in 9%. There were three on-study deaths (i.e. occurring &lt;30 days after last dose). Conclusions US MM-6 patients reflect the heterogeneous real-world US MM population; the population for this study includes patients from the community who may not be eligible for traditional clinical trials. These updated data in mostly elderly, comorbid, NDMM patients treated in the community setting demonstrate the feasibility and tolerability of iCT to IRd after 3 cycles of bortezomib-based induction; approximately half of patients remain on treatment, and enrollment is ongoing. iCT to IRd resulted in improved responses, with increased rates of ≥VGPR, and prolonged DOT and may thereby improve outcomes for real-world patients. iCT to an all oral regimen could also prevent treatment interruptions for patients who are unable to or prefer not to travel in the context of travel restrictions or other factors. Disclosures Girnius: Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Yimer:TG Therapeutics: Consultancy; AstraZeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company), Speakers Bureau; Sanofi: Speakers Bureau; Texas Oncology: Current Employment; BeiGene: Other: TRAVEL, ACCOMODATIONS, EXPENSES (paid by any for-profit health care company), Research Funding, Speakers Bureau; Janssen: Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company), Research Funding, Speakers Bureau; Takeda: Speakers Bureau; Celgene, a Bristol-Myers Squibb Company: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company), Speakers Bureau; Karyopharm: Consultancy, Divested equity in a private or publicly-traded company in the past 24 months, Membership on an entity's Board of Directors or advisory committees, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company), Speakers Bureau; Epizyme: Consultancy, Divested equity in a private or publicly-traded company in the past 24 months. Noga:Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Current Employment. Manda:AbbVie: Other: Investigator in AbbVie-sponsored clinical trials. Lyons:Texas Oncology/US Oncology: Current Employment; Novartis: Honoraria. Bogard:Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Current Employment. Whidden:Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Current Employment. Cherepanov:Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Current Employment. Lu:Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Current Employment. Aiello:Takeda: Honoraria; Travera: Honoraria; Celgene: Honoraria; Karyopharm: Honoraria. Richter:Celgene: Consultancy, Speakers Bureau; Adaptive Biotechnologies: Consultancy, Speakers Bureau; Janssen: Speakers Bureau; Sanofi: Consultancy; Karyopharm: Consultancy; Takeda: Consultancy; AstraZeneca: Consultancy; Secura Bio: Consultancy; Bristol Myers Squibb: Consultancy; X4 Pharmaceuticals: Consultancy; Oncopeptides: Consultancy; Antengene: Consultancy. Rifkin:McKesson: Current equity holder in publicly-traded company, Ended employment in the past 24 months, Other: Stock ownership; Takeda, Amgen, Celgene, BMS, Mylan, Coherus BioSciences, Fresenius: Consultancy; AbbVie: Other: Investigator in AbbVie sponsored clinical trials; Takeda, Amgen, BMS (Celgene): Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: Real-world evaluation of long-term proteasome inhibition with ixazomib in combination with lenalidomide and dexamethasone for the treatment of newly diagnosed multiple myeloma in non-transplant patients with stable disease after 3 cycles of a bortezomib-based induction.

This thesis describes the development of a computer controlled on-line system for fermentation monitoring and control. The entire system consists of a laboratory fermenter, flow injection system (four channels), a newly designed on-line filter, biomass analysis channel, pH and oxygen controllers as well as a spectrophotometer. A new design of gas driven flow injection analysis (FIA) allows a large number of reagents to be handled. The computer-controlled four channel PIA system is well suited for sequential analysis, which is important for fermentation on-line mOnitoring. The system can change the wavelength of the spectrophotometer automatically for each PIA channel, which makes the system powerful and flexible. A high frequency, low energy ultrasonic filter was modified and applied to the system for on-line mammalian cell culture sampling without breaking the sterile barrier. The results show that this novel application of ultrasonic filter technology results in higher efficiency and reliability and a longer life cycle than other types of filter. All the operations of the analytical system are controlled by a Macintosh computer (Quadra 950). The control program was written in LabVIEW which is a graphical programming language and well applicable to fermentation control. The software communicates with detectors, data acquisition, data analysis and presentation. The system can programmatically control up to 50 devices. Mammalian cell batch culture was used as an example of the application of the system. The system consists of a laboratory fermenter with a continuous sample withdrawal filter and an analysis system where glucose, lactate and ammonia, lactate dehydrogenase and biomass were measured. Cell viability was estimated by microscopic assay with trypan blue. pH and Oxygen were also measured. The system response was fast and yields a large number of reliable and precise analytical results which can be of great importance in the monitoring and control of mammalian cell culture conditions.

The lens of the eye needs to be completely transparent in order to allow all light entering the eye to reach the retina. This transparency is maintained by the highly ordered structure of the lens proteins the crystallins. Any disruption to the lens proteins can cause an opacity to develop which is known as cataract. During cortical cataract formation there is increased truncation of the lens crystallins. It is believed that overactivation of calcium-dependent cysteine proteases, the calpains, is responsible for the increased proteolysis of the crystallins seen during cataractogenesis. Within the ovine lens there are three calpains, calpain 1, 2 and the lens specific calpain Lp82. The aim of this thesis was to determine the changes in the lens proteins during ageing and cataractogenesis, and to establish the role of the calpains in these processes. Calpain 1 and 2 were purified from ovine lung and Lp82 was purified from lamb lenses using chromatography. Activity and presence of the calpains was determined by using the BODIPY-FL casein assay, gel electrophoresis, Western blot and casein zymography. Changes in the lens proteins, specifically the crystallins, were visualised using two-dimensional electrophoresis (2DE). Lenses from fetal, 6 month old and 8 year old sheep were collected, as well as stage 0, 1, 3 and 6 cataractous ovine lenses. The proteins from the lenses were separated into the water soluble and urea soluble fractions and analysed by 2DE. Mass spectrometry was used to determine the masses and therefore modifications of the crystallins. Finally, the individual crystallins were separated using gel filtration chromatography and incubated with the purified calpains in the presence of calcium. The extent of the proteolysis was visualised using 2DE and truncation sites determined by mass spectrometry. Purification of the calpains resulted in samples that were specific for each calpain and could be used in further experiments. 2DE analysis showed that there were changes to the crystallins during maturation of the lens. The α-crystallins become increasingly phosphorylated as the lens ages and a small amount becomes truncated. The β-crystallins were also modified during ageing by truncation and deamidation. When crystallins from cataractous lenses were compared using 2DE there were changes to both the α- and β-crystallins. The α-crystallins were found to be extensively truncated at their C-terminal tail. Four of the seven β-crystallins, βB1, βB3, βB2 and βA3, showed increased truncation of their N-terminal extensions during cataract formation. All three calpains truncated αA and αB-crystallin at their C-terminal ends after incubation. Calpain 2 and Lp82 each produced unique αA-crystallin truncations. All three calpains truncated βB1 and βA3 and calpain 2 also truncated βB3. When the truncations from the calpain incubations were compared to those seen during cataract formation, many of the truncations were found to be similar. Both the unique truncations from calpain 2 and Lp82 were found in cataractous lenses, with the Lp82 more obvious in the 2DE. The β-crystallin truncations found after incubation with the calpains were similar to those found during cataractogenesis. In conclusion this study documents the changes to the ovine lens during maturation and cataractogenesis and indicates a role for the calpain family in the increased proteolysis observed in the ovine cataract.

Methods for the preparation of concentrates of factor VIII, factor IX, high purity factor IX, Cl esterase inhibitor, specific immunoglobulin and platelet factor XIII are described. These procedures were developed or modified with the aim of integration into an automated process that would allow sequential recovery of all the clinically significant trace proteins from a single plasma pool. Concomitant recovery of important proteins such as transferrin, alpha-1-antitrypsin and platelet-derived growth factor was considered. A high-purity factor VIII concentrate heat-treated at 80°C for 96 h was prepared by a process that incorporated heparin fractionation. This method was shown to be suitable for assimilation into an existing regional blood processing laboratory. Several ion-exchange procedures for the recovery of factor IX were evaluated and higher purification of a factor IX concentrate was achieved on a new cellulose-based chromatographic medium. A chromatographic procedure for the preparation of a heat-treated high-purity Cl esterase inhibitor concentrate was described and the performance of a new cellulose-based desalting medium was evaluated in comparison with ultrafiltration. A heat-treated specific immunoglobulin concentrate was prepared from side-stream fractions of an automated chromatographic process for the production of albumin concentrate, and a pilot study for the fractionation of outdated platelet concentrates was carried out with the aim preparing components of potential therapeutic value. See summary flow diagrams of fractionation processes included with references in the back of this thesis.

External representations (ERs) used in science education are multimodal ensembles consisting of design elements to convey educational meanings to the audience. As an example of a dynamic ER, an animation presenting its content features (i.e., scientific concepts) via varying the feature’s depiction over time. A production team invited the dissertation author to inspect their creation of a biochemistry animation about the role of the actin cytoskeleton in cell motility and the animation’s implication on learning. To address this, the author developed a four-step methodology entitled the Multimodal Variation Analysis of Dynamic External Representations (MVADER) that deconstructs the animation’s content and design to inspect how each content feature is conveyed via the animation’s design elements.

This dissertation research investigated the actin animation’s educational value and the MVADER’s utility in animation evaluation. The research design was guided by descriptive case study methodology and an integrated framework consisting of the variation theory, multimodal analysis, and visual analytics. As stated above, the animation was analyzed using MVADER. The development of the actin animation and the content features the production team members intended to convey via the animation were studied by analyzing the communication records between the members, observing the team meetings, and interviewing the members individually. Furthermore, students’ learning experiences from watching the animation were examined via semi-structured interviews coupled with post- storyboarding. Moreover, the instructions of MVADER and its applications in studying the actin animation were reviewed to determine the MVADER’s usefulness as an animation evaluation tool.

Findings of this research indicate that the three educators in the production team intended the actin animation to convey forty-three content features to the undergraduate biology students. At least 50% of the student who participated in this thesis learned thirty-five of these forty-three (> 80%) features. Evidence suggests that the animation’s effectiveness to convey its features was associated with the features’ depiction time, the number of identified design elements applied to depict the features, and the features’ variation of depiction over time.

Additionally, one-third of the student participants made similar mistakes regarding two content features after watching the actin animation: the F-actin elongation and the F-actin crosslink structure in lamellipodia. The analysis reveals the animation’s potential design flaws that might have contributed to these common misconceptions. Furthermore, two disruptors to the creation process and the educational value of the actin animation were identified: the vagueness of the learning goals and the designer’s placement of the animation’s beauty over its reach to the learning goals. The vagueness of the learning goals hampered the narration scripting process. On the other hand, the designer’s prioritization of the animation’s aesthetic led to the inclusion of a “beauty shot” in the animation that caused students’ confusion.

MVADER was used to examine the content, design, and their relationships in the actin animation at multiple aspects and granularities. The result of MVADER was compared with the students’ learning outcomes from watching the animation to identify the characteristics of content’s depiction that were constructive and disruptive to learning. These findings led to several practical recommendations to teach using the actin animation and create educational ERs.

To conclude, this dissertation discloses the connections between the creation process, the content and design, and the educational implication of a biochemistry animation. It also introduces MVADER as a novel ER analysis tool to the education research and visualization communities. MVADER can be applied in various formats of static and dynamic ERs and beyond the disciplines of biology and chemistry.

Prostate cancer is (PCa) the second leading cause of cancer death in males in the United State, with 174,650 new cases and 31,620 deaths estimated in 2019. Polo-like kinase 1 (PLK1) has been postulated to have a pro-tumorigenesis function, besides its critical role in regulation of cell cycle, and to be overexpressed in various types of human cancer, including prostate cancer (PCa). However, our understanding remains unclear regarding the pro-tumor properties of PLK1 partially due to a lack of proper animal model. Integrating our recently generated prostate-specific PLK1 knock-in genetically engineered mouse model (GEM) and the transcriptome data of human PCa patients, we identify an oncogenic role of PLK1 in the prostate adenocarcinoma progression, castration resistance and metastatic dissemination. To elucidate the underlying mechanism, we investigate the link between PLK1 and tumor microenvironment in PCa using the transgenic mouse model, and find that PLK1overexpression enable the macrophages polarization towards M2 phenotype via driving the activation of IL4/IL13/STAT6 pathway. These findings first validates PLK1 as a critical oncogene closely associated with PCa progression in vivo, and uncover a novel function of PLK1 to facilitate IL4/STAT6 signaling and M2 macrophage polarization. Importantly, these findings suggest an efficient therapeutic strategy targeting STAT6 for treatment of advanced PCa which usually possessing a high level of PLK1 expression. To further explore the molecular mechanism underlying PLK1-induced PCa progression and resistance to therapy, we turned our eyes to epigenetic modifications. It has been documented that epigenetic deregulation such as histone modification and DNA methylation contributes to PCa initiation and progression. Enhancer of zeste homologue 2 (EZH2), the catalytic subunit of Polycomb-repressive complex 2 (PRC2), plays a critical role in repressing gene expression by tri-methylation of histone 3 at lysine 27 (H3K27me3). Emerging data have demonstrated that there is a link between EZH2 and oncogenesis as EZH2-mediated methylation acts as an important factor in epigenetic silencing of tumor suppressor genes in cancer. Expression of EZH2 is often upregulated in castration-resistant prestate cancer (CRPC), thus EZH2 has been proposed as a target for CRPC. Importantly, it has been demonstrated that EZH2 becomes hyperphosphorylated in CPRC cells. Further, it has been shown that the oncogenic function of EZH2 is usually regulated by the post-translational modifications. PLK1 acting as a serine/threonine kinase to regulate multiple signaling pathways in human cancer, however, whether PLK1 is involved in EZH2 phosphorylation is not known. Herein, we show that Plk1 physically interacts with EZH2 and negatively regulates H3K27 trimethylation (H3K27me3). Furthermore, Plk1 can phosphorylate EZH2 at T144, and Plk1-mediated phosphorylation of EZH2 is involved in inhibiting EZH2 activity toward H3K27me3. More importantly, EZH2 phosphorylation by Plk1 is inhibitory for PRC2-mediated gene repression but required for transcriptional activation toward oncogenesis. Finally, by combination with Plk1 inhibitor BI2536, we show a robust sensitization of EZH2 inhibitors in CRPC cell lines, as well as in CRPC xenograft tumors. Our findings provide a new mechanism to define the oncogenic activity of EZH2 and suggest that inhibition of Plk1-mediated EZH2 activity may provide a promising therapeutic approach for CRPC.

The phenylpropanoid pathway gives rise to a wide variety of specialized metabolites, but the majority of carbon flux going through this pathway is directed towards the synthesis of the lignin monomers: p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol. Lignin is a major impediment in biomass saccharification, which negatively affects animal feed and biofuel production. In an effort to improve biomass for the latter purposes, researchers have altered the polymer through genetic manipulations and generated biomass with lower recalcitrance to saccharification; however, in many cases these efforts have resulted in plant dwarfism. To date, we do not have a full understanding of the extent of lignin modifications a plant is able to tolerate without affecting its growth. More importantly, the mechanism that links dwarfism and modifications in lignin content and composition remains unknown. To contribute to answering these questions, we designed a strategy to incorporate a novel monomer into the lignin of Arabidopsis thaliana. We used mutants in genes that code for enzymes and regulators of the phenylpropanoid pathway to redirect the pathway’s flux towards the synthesis of p-coumaraldehyde and prevent the incorporation of p-coumaryl alcohol. Despite being mutated for the genes typically considered to be required for monolignol biosynthesis, the plants we generated continue to incorporate p-coumaryl alcohol into their lignin. This result suggests that the pathway’s architecture has not been completely elucidated and that there are more enzymes involved in lignification than previously thought. Additionally, we explored the connection between perturbations in phenylpropanoid metabolism and plant growth, by using an inducible system to track the changes in gene expression and metabolism that occur when phenylpropanoid metabolism is restored in a lignin biosynthetic mutant. The use of an inducible system allowed us to not only determine the metabolic processes affected in this mutant, but the proximal sequence of events that lead to restored growth when a functional copy of the mutant gene is induced. Finally, we redirected the flux through the pathway to assess the effects of simultaneously modulating lignin content and composition. Through this project we discovered that redirecting phenylpropanoid flux towards the synthesis of sinapyl alcohol in lignin-deficient mutant backgrounds, results in plant dwarfism. The growth impairment of these mutants can be overcome by providing exogenous coniferyl alcohol, suggesting that dwarfism in these mutants is caused by deficiency in coniferyl alcohol and/or derivatives thereof and not lignin alone. Altogether these projects allowed us to define the cellular processes affected by perturbations in phenylpropanoid homeostasis and the role of other phenylpropanoids besides lignin in this process.

Malonyl-thioesters are reactive at the thioester carbonyl and the carboxylate moieties, as seen in acyl transfer or hydrolysis and decarboxylation. Enzymes use these reactive centers to perform different enzyme chemistry throughout metabolism. This enzyme chemistry coupled with the inherent reactivity of malonyl-thioesters makes structure-function studies difficult. When malonyl-thioesters are used for structure-function studies, it usually results in a hydrolyzed or decarboxylated product. There are examples, however, where this is overcome, many of which are discussed throughout this thesis. To overcome the inherent reactivity of malonyl-thioesters and enzymes, analogs have been synthesized to perform structure-function studies. Initial studies focused on altering the thioester carbonyl to limit hydrolysis and decarboxylation; however, these studies revealed the importance of retaining the thioester carbonyl to be positioned in the oxyanion hole. My thesis work focused on the synthesis, characterization, and use in structure-function studies of malonyl-thioester analogs that either preserve the thioester carbonyl or alter it to an ester or amide, and alter the carboxylate to a sulfonate or nitro group. After synthesizing the methylmalonyl-CoA analogs, we performed structure-function studies with methylmalonyl-CoA decarboxylase. This case study revealed the potential of these analogs to both inhibit decarboxylase activity and their use in structure-function studies to gain mechanistic insights. This successful study prompted us to continue these structure-function studies in enzymes with different chemistries such as an epimerase or bi-functional acyltransferase/decarboxylase. The widespread use of these methylmalonyl-CoA analogs also motivated us to add more malonyl-thioester analogs to our toolbox. I have preliminary data that these malonyl-thioester analogs inhibit β-keto-acyl-synthase III, an enzyme involved in fatty acid production in E. coli. This inhibition gives us confidence that these analogs will be useful in structure-function studies that will reveal answers to long standing mechanism and protein-protein interaction questions in the polyketide and fatty acid synthase field.

Viruses are pathogenic agents which affect all varieties of organisms, including plants, animals and humans. These microscopic particles are genetically simple organisms which encode a limited number of proteins that undertake a wide range of functions. While structurally distinct, viruses often share common characteristics that have evolved to aid in their infectious life cycles. A commonly underappreciated characteristic of many deadly viruses is a lipid envelope coat that surrounds them. Lipid enveloped viruses comprise a diverse range of pathogenic viruses, known to cause disease in both animals and human which often leads to high fatality rates, many of which lack effective and approved therapeutics. This report focuses on learning how a multifunctional protein within lipid enveloped viruses, the matrix protein, interacts with the plasma membrane of cells to enter and exit cells. Specifically, four viruses are investigated, Measles virus and Nipah virus (within the Paramyxoviridae family) and Ebola virus and Marburg virus (within the Filoviridae family). Through numerous in vitro experiments, functional cellular assays, a myriad of microscopy techniques, and experiments in high containment bio-safety level 4 settings, this report identifies specific lipids at play during the viral assembly process for each virus. Moreover, mechanistic insight is presented as to how each matrix protein interacts with the plasma membrane to facilitate: membrane association, viral matrix protein oligomerization and assembly, the rearrangement of lipids within the plasma membrane, and viral production. Lastly, numerous small molecule inhibitors targeting specific lipids, (e.g. phosphatidylserine and phosphatidylinositol 4,5 bisphosphate) within the cell were investigated for their efficacy in inhibiting matrix protein-dependent viral like particle production and viral spread in cells. As a whole, these projects lend credence to the significant role that lipids and the plasma membrane play throughout lipid enveloped viral life cycles, and provide compelling evidence for the merit of future drug-development research geared at targeting the matrix protein-plasma membrane interaction.

Accurate DNA replication is vital for maintaining genomic stability. Consequently, the machinery required to drive this process is designed to ensure the meticulous maintenance of information. However, random misincorporation of errors reduce the fidelity of the DNA and lead to pre-mature aging and age-related disorders such as cancer and neurodegenerative diseases. Some of the incorporated errors are the result of the error prone DNA polymerase alpha (Pol a), which initiates synthesis on both the leading and lagging strand. Lagging strand synthesis acquires an increased number of polymerase a tracks because of the number of Okazaki fragments synthesized per round of the cell cycle (~50 million in mammalian cells). The accumulation of these errors invariably reduces the fidelity of the genome. Previous work has shown that these pol a tracks can be removed by two redundant pathways referred to as the short and long flap pathway. The long flap pathway utilizes a complex network of proteins to remove more of the misincorporated nucleotides than the short flap pathway which mediates the removal of shorter flaps. Lysine acetylation has been reported to modulate the function of the nucleases implicated in flap processing. The cleavage activity of the long flap pathway nuclease, Dna2, is stimulated by lysine acetylation while conversely lysine acetylation of the short flap pathway nuclease, FEN1, inhibits its activity. The major protein players implicated during Okazaki fragment processing (OFP) are known, however, the choice of the processing pathway and its regulation by lysine acetylation of its main players is yet unknown. This dissertation identifies three main findings: 1) Saccharomyces cerevisiae helicase, petite integration frequency (Pif1) is lysine acetylated by Esa1 and deacetylated by Rpd3 regulating its viability and biochemical properties including helicase, binding and ATPase activity ii) the single stranded DNA binding protein, human replication protein A (RPA) is modified by p300 and this modification stimulates its primary binding function and iii) lysine acetylated human RPA directs OFP towards the long flap pathway even for a subset of short flaps.