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Update This article was updated on January 14, 2020, because of a previous error. On page 1, in the Abstract and the section entitled “Case Reports,” the word that had read “Genentech“ now reads “Genetech.” An erratum has been published: JBJS Case Connect. 2020 Feb 5;10(1):e0363ER. Case: The use of biologics is rapidly expanding. Over the past decade, there has been a significant increase in the number of cash-based “stem cell”/regenerative medicine clinics in the United States. These clinics provide cash-based services touting stem cell injections to cure a myriad of conditions. Largely, these clinics are unregulated and using injections in a non–Food and Drug Administration–approved manner. We report on 2 patients who presented with symptoms suggestive of septic arthritis following stem cell injections by cash-based local stem cell clinics. Case 1 involved a patient who developed septic arthritis following an injection of umbilical cord blood–derived cellular products (Genetech) and required an antibiotic spacer followed by a total hip arthroplasty. Case 2 involved a patient who developed a likely immune-mediated reaction following an injection of morselized human placental allograft tissue by a local chiropractic office at a cost of approximately $8,000. Conclusions: We present these cases to bring increased awareness to the issue and call for increased regulation of this practice.

AbstractOne of the hallmarks of aging is the progressive accumulation of senescent cells in organisms, which has been proposed to be a contributing factor to age‐dependent organ dysfunction. We recently reported that Bruton's tyrosine kinase (BTK) is an upstream component of the p53 responses to DNA damage. BTK binds to and phosphorylates p53 and MDM2, which results in increased p53 activity. Consistent with this, blocking BTK impairs p53‐induced senescence. This suggests that sustained BTK inhibition could have an effect on organismal aging by reducing the presence of senescent cells in tissues. Here, we show that ibrutinib, a clinically approved covalent inhibitor of BTK, prolonged the maximum lifespan of a Zmpste24−/− progeroid mice, which also showed a reduction in general age‐related fitness loss. Importantly, we found that certain brain functions were preserved, as seen by reduced anxiety‐like behaviour and better long‐term spatial memory. This was concomitant to a decrease in the expression of specific markers of senescence in the brain, which confirms a lower accumulation of senescent cells after BTK inhibition. Our data show that blocking BTK has a modest increase in lifespan in Zmpste24−/− mice and protects them from a decline in brain performance. This suggests that specific inhibitors could be used in humans to treat progeroid syndromes and prevent the age‐related degeneration of organs such as the brain.

Background: Unlike heart failure with reduced ejection fraction, there is no approved treatment for heart failure with preserved ejection fraction, the predominant phenotype in women. Therefore, there is a greater heart failure therapeutic deficit in women compared with men. Methods: In a prespecified subgroup analysis, we examined outcomes according to sex in the PARAGON-HF trial (Prospective Comparison of ARNI With ARB Global Outcomes in Heart Failure With Preserved Ejection Fraction), which compared sacubitril-valsartan and valsartan in patients with heart failure with preserved ejection fraction. The primary outcome was a composite of first and recurrent hospitalizations for heart failure and death from cardiovascular causes. We also report secondary efficacy and safety outcomes. Results: Overall, 2479 women (51.7%) and 2317 men (48.3%) were randomized. Women were older and had more obesity, less coronary disease, and lower estimated glomerular filtration rate and NT-proBNP (N-terminal pro–B-type natriuretic peptide) levels than men. For the primary outcome, the rate ratio for sacubitril-valsartan versus valsartan was 0.73 (95% CI, 0.59–0.90) in women and 1.03 (95% CI, 0.84–1.25) in men ( P interaction = 0.017). The benefit from sacubitril-valsartan was attributable to reduction in heart failure hospitalization. The improvement in New York Heart Association class and renal function with sacubitril-valsartan was similar in women and men, whereas the improvement in Kansas City Cardiomyopathy Questionnaire clinical summary score was less in women than in men. The difference in adverse events between sacubitril-valsartan and valsartan was similar in women and men. Conclusions: As compared with valsartan, sacubitril-valsartan seemed to reduce the risk of heart failure hospitalization more in women than in men. Whereas the possible sex-related modification of the effect of treatment has several potential explanations, the present study does not provide a definite mechanistic basis for this finding. Clinical Trial Registration: https://www.clinicaltrials.gov . Unique identifier: NCT01920711.

Abstract Introduction. Growing evidence suggests that immune cells that reside within the tumor microenvironment are dysregulated and functionally impaired, leading to defective anti-tumor immunity of the host. One of the major immunosuppressive mechanisms during tumor progression is expansion of regulatory immune cells. Here, we analyzed the immune cells within the bone marrow (BM) and the peripheral blood (PB) of 2 immunocompetent multiple myeloma (MM) mouse models. We next studied the role of regulatory T cells (Tregs) in MM pathogenesis. Materials and methods. To study the immune cell populations of the BM and PB, we used two immuncompetent mouse models and transplanted VK*MYC cells or 5TGM1 cells into C57BL/6 and C57BL/Kalwrij mice respectively. The immune cell populations and checkpoint receptor expressions were analyzed by CyTOF mass cytometer or flow-cytometry. Treg induction assay was performed in vitro to study the mechanism of Treg increase in the BM of myeloma injected mice. CD4+ CD25- cells were obtained from C57BL/Kalwrij mice and were co-cultured with 5TGM1 cells or B cells from C57BL/Kalwrij mice in vitro. Treg induction was compared by flow-cytometry. Transplantable VK*MYC cells were injected into "depletion of regulatory T cell" (DEREG) mice, which expresses a simian diphtheria toxin (DT) receptor-enhanced GFP fusion protein under the control of the FOXP3 gene locus, or their wild type littermates. DT injection into these mice leads to depletion of Tregs as previously described (J Exp Med. 2007; 204: 57-63). DT was given once every week for a total 3 times i.p to the DEREG mice or the littermate controls to specifically deplete Tregs and to study the role of Tregs during MM progression. Tregs (CD4+ FOXP3-GFP+ cells) were sorted from VK*MYC injected mice or non-injected DEREG mice BM using FACSAria cell sorter. Cells isolated were subjected to RNA sequencing. Gene Set Enrichment Analysis (GSEA) was performed to define differences in molecular signatures between MM-associated and normal Tregs. Results. The Treg proportion was significantly increased within the CD4+ T cells in the BM of myeloma cell injected mice from the early stage of disease compared to control mice, while in the PB, the increase was observed only at the late stages of disease progression. The effector T cell (Teff)/Treg ratio was significantly decreased in the BM at the end-stage myeloma bearing mice (P<0.01). Checkpoint related molecules (PD-1, LAG-3 and Tim-3) on Tregs and Teffs were up-regulated at the protein level in the BM of myeloma injected mice compared to control (P<0.01). These data indicate the activation of Tregs and decrease of Teff activity, leading to suppression of anti-myeloma T cell activity in the MM BM. Under in vitro co-culture conditions, 5TGM1 cells induced a significant increase in the number of Tregs from non-Treg CD4+ T cells compared to controls (P<0.01). 5TGM1 cell-induced Tregs presented with enhanced ki-67 expression, thus suggesting the ability of MM cells to induce Treg proliferation. Additionally, the trans-well co-culture experiment showed that the major mechanism for Treg induction by 5TGM1 was by direct contact with T cells rather than secreting factors. Significant increase in survival was observed in the VK*MYC injected DEREG mice under Treg depletion compared to the DEREG mice without Treg depletion and wild type littermates under DT treatment (P<0.001). The Treg depleted DEREG mice were accompanied with an increase of Teffs (P<0.01), indicating recovery of anti-myeloma T cell activity. RNA sequencing of BM Tregs from VK*MYC injected mice showed increased expression of immune checkpoint related molecules and increased Treg effector molecules (IL-10, Granzyme B) compared to BM Tregs from control mice, indicating a more functionally active phenotype of VK*MYC associated Tregs. GSEA showed an enrichment of genes involved in type-1 interferon signaling in VK*MYC associated Tregs. Conclusions. We used 2 immunocompetent MM mouse models to study the characterization and significance of Tregs in MM progression. CyTOF analysis and RNA sequencing data indicated Treg activation in the MM BM microenvironment. The Treg in vivo depletion experiment showed that Tregs have a significant role in MM progression. These data indicate that immunotherapy targeting Tregs may represent a novel therapeutic strategy for MM. Studies are ongoing to understand the roles of Tregs in human MM patients. Disclosures No relevant conflicts of interest to declare.

Background Bortezomib (BTZ) is highly effective in the treatment of multiple myeloma; however, emergent drug resistance is common. The underlying mechanisms of such proteasome inhibitor resistance are still incompletely understood. Methods To further understand its resistant mechanism, we generated eight multiple myeloma (MM) cell lines resistant to bortezomib (BTZ) by exposure to increasing drug concentration: five of them acquired novel PSMB5 mutations. Given the rarity of similar mutations in over 1,500 analyzed MM patients, we explored in depth the role of the proteasome on MM cell viability and BTZ sensitivity by systematically deleting the major proteasome targets of BTZ by CRISPR. Results We demonstrated that MM cell lines without PSMB5 were surprisingly viable (mutation corresponding yeast gene pre2 is lethal). PSMB5 mutated, BTZ resistant, MM cell lines were re-sensitized to BTZ when PSMB5 was experimentally deleted, implying that this mutation is activating in its drug resistance function. In contrast PSMB6 knockout was lethal to MM cell lines, which were efficiently rescued by re-introduction of wild type PSMB6. Interestingly, reduction in PSMB6 levels also prevented the splicing of the major catalytic subunits PSMB5, PSMB7, PSMB8 and PSMB10. PSMB6 engineered with no splicing function or catalytic activity, also restored viability, inferring that the contribution of PSMB6 to proteasome structure is more important than functional activity. Supporting this observation, BTZ sensitivity was restored in resistant MM cells line by introducing low level expression of mutated PSMB6 lacking splicing function. As with PSMB6, PSMB7 knockout was lethal to MM cell lines. In contrast, loss of immunoproteasome subunits PSMB8 and PSMB9 was neither lethal nor restored sensitivity to BTZ. Our results demonstrate that expression of the three constitutive proteasome subunits PSMB5, PSMB6 and PSMB7 is highly co-dependent. This dependence is relying on the structure, but not the function, of PSMB5 and PSMB6. Conclusions In summary, PSMB5 and PSMB6, but not PSMB8 and PSMB9, are highly relevant for BTZ sensitivity in MM. Absence of PSMB6 or PSMB7, but not PSMB5, was lethal in MM cell lines. Expression of PSMB5, PSMB6 and PSMB7 was highly co-dependent. Together these findings suggest that the modulation of expression rather than function of PSMB5, PSMB6 or PSMB7 may be a new therapeutic strategy. Disclosures No relevant conflicts of interest to declare.

Background: The 2018 iwCLL guidelines recommend close observation of early stage CLL pts who do not meet indications for therapy ("watch and wait" strategy). Prior attempts at early therapeutic intervention in unselected early-stage CLL pts using alkylating agents such as chlorambucil and chemoimmunotherapy failed to show a significant benefit; and these therapies were associated with substantial toxicities. The introduction of Bruton tyrosine kinase (BTK) inhibitors has revolutionized the treatment landscape of CLL. The German CLL Study group recently presented results of the CLL12 study which demonstrated a significant improvement in event-free survival (EFS) among untreated early stage high risk CLL pts who received ibrutinib compared to placebo (median EFS not reached vs. 47.8 months, respectively; Langerbeins, EHA, 2019). Acalabrutinib is a more selective second-generation BTK inhibitor that has fewer adverse events in pts with CLL, and has shown equally impressive clinical activity for CLL pts. We are conducting an investigator initiated randomized phase 2 study to compare acalabrutinib with or without obinutuzumab (a glycoengineered anti-CD20 monoclonal antibody approved in the treatment of CLL) among high risk early stage CLL pts. Study Design: All pts with newly diagnosed (<2 years from registration) previously untreated early stage CLL who do not meet the 2018 iwCLL guidelines for initiation of therapy are eligible for enrollment in this study. Pts undergo risk stratification according to the CLL-International prognostic index (CLL-IPI) which consists of the following 5 variables: age >65 years (1 point), Rai stage I-IV (1 point), del17p and/or TP53 mutation (4 points), unmutated immunoglobulin heavy chain (IGHV) genes (2 points), and serum β2-microglobulin >3.5 g/dL (2 points). Pts with high (4-6) and very high (7-10) risk CLL-IPI are randomly (1:1) assigned to acalabrutinib 100 mg orally twice daily (Arm A) or acalabrutinib with obinutuzumab at a standard approved schedule (Arm B). Treatment with acalabrutinib is continued for a minimum of 2 years unless the patient has unacceptable side effects or withdraws consent. Pts with low (0-1) and intermediate (2-3) risk CLL-IPI score are assigned to an observation arm with follow-up once every 6 months for 2 years, and then according to routine clinical practice (Arm C). Endpoints: The primary endpoint of the intervention arms (Arms A and B) is the achievement of minimal residual disease (MRD)-negative complete remission in the bone marrow after 2 years of therapy. MRD is assessed using an 8-color flow cytometry with a detection limit of 0.01%. The primary endpoint of the observation arm (Arm C) is time to first therapy. Secondary endpoints for all arms are: progression-free survival, overall survival, and safety. Statistical design: A randomized trial comparing the experimental arm (acalabrutinib plus obinutuzumab) against the control arm (acalabrutinib alone) will be conducted as described by Rubinstein. A sample size of 36 pts per arm provides 80% power to detect an improvement in MRD-negative complete response rate from 10% to 30%, using a one-sided test at a significance level of 0.10 (EAST 6.4). We anticipate accruing an additional 8 high/very high risk pts (4 in each arm) to account for ineligibility, cancellation, or major treatment violation. The total enrollment for Arms A and B will be 80 pts. We will enroll 40 pts to Arm C; for a total enrollment of 120 pts. Key correlatives: Comprehensive profiling to assess the innate and adaptive immune system in paired peripheral blood and bone marrow will be conducted in all pts registered to Arms A and B at baseline, 12 and 24 months after enrollment. In addition, bone marrow hematopoietic function will be assessed in all pts at these time points. We will employ a CLL focused custom 61-gene panel to estimate tumor mutational burden as well as mutational profiles for each patient at baseline, 12, and 24 month time points, as well at the time of disease progression. Preliminary results: The trial is registered at clinicaltrials.gov NCT03516617. The trial opened to accrual at all three Mayo Clinic sites (Rochester, MN; Jacksonville, FL; and Scottsdale, AZ) in September 2018. Twenty-eight pts have been registered; the baseline characteristics are shown in Table 1. The trial is expected to complete enrollment in September 2020; and the primary analysis will be available in September 2022. Disclosures Parikh: Janssen: Research Funding; AstraZeneca: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; AbbVie: Honoraria, Research Funding; Acerta Pharma: Research Funding; Ascentage Pharma: Research Funding; Genentech: Honoraria. Ding:DTRM Biopharma: Research Funding; Merck: Research Funding. Ailawadhi:Pharmacyclics: Research Funding; Cellectar: Research Funding; Takeda: Consultancy; Amgen: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Celgene: Consultancy. Kenderian:Novartis: Patents & Royalties, Research Funding; Tolero: Research Funding; Humanigen: Other: Scientific advisory board , Patents & Royalties, Research Funding; Lentigen: Research Funding; Morphosys: Research Funding; Kite/Gilead: Research Funding. Chanan-Khan:Mayo Clinic: Employment; Ascentage: Research Funding; Millennium: Research Funding; Jansen: Research Funding; AbbVie: Research Funding; Xencor: Research Funding; Pharmacyclics: Research Funding; Merck: Research Funding. Kay:Infinity Pharmaceuticals: Other: DSMB; Celgene: Other: Data Safety Monitoring Board; Agios: Other: DSMB; MorphoSys: Other: Data Safety Monitoring Board. OffLabel Disclosure: Drug: Acalabrutinib Purpose: treatment of CLL

Background: The 2008 International Working Group for CLL (IWCLL) criteria define MBL as a clonal B-cell disorder with a peripheral blood B-cell count of <5 x 109/L, in the absence of cytopenias(s), lymphadenopathy and/or organomegaly on physical examination, and no other concomitant lymphoproliferative disorder. In the presence of palpable lymphadenopathy and/or organomegaly, and a B-cell count of < 5 x 109/L, a diagnosis of small lymphocytic lymphoma (SLL) is established. A study by Gentile et al (Am J Hematol. 2013) reported that 29 out of 62 individuals with clinically identified MBL (42%) had lymphadenopathy identified on CT imaging. Notably, however, after a median follow-up of 35 months, the rate of progression among cases of MBL with lymphadenopathy identified on CT imaging was only 6.9% (2/29 patients), which was no different than in cases of MBL without lymphadenopathy on CT imaging (rate of progression, 9%; 3/33 patients). Our study sought to 1) determine if lymphadenopathy and/or organomegaly detected by imaging studies performed for unrelated reasons in individuals with MBL can identify a subset with shorter time to first CLL therapy (TFT) and overall survival (OS); and 2) compare outcomes of individuals with MBL who have incidentally detected lymphadenopathy/splenomegaly and SLL patients. Methods: We used the Mayo Clinic CLL Database to identify individuals with high-count MBL (absolute B-cell count: 0.5 - 4.9 x 109/L) and divided them into the following three cohorts: a) Cohort A: no imaging studies available at MBL diagnosis; b) Cohort B: imaging studies done within one year of MBL with no evidence of lymphadenopathy and/or organomegaly; and c) Cohort C: imaging studies done within one year of MBL with lymphadenopathy and/or organomegaly, attributable to MBL. All individuals with MBL were first seen at Mayo Clinic after 1/1/2002; those with a concomitant lymphoproliferative disorder within 3 months of diagnosis were excluded. Patients with SLL seen during the same time interval were identified as a comparison cohort. We compared the baseline characteristics (Kruskal Wallis for continuous and Chi-square for categorical variables) and TFT and OS across the three MBL cohorts, and patients with SLL. Cumulative incidence of TFT was adjusted for the competing risk of death. Age- and sex-adjusted OS was analyzed using inverse weights methods. Results: 657 individuals with high-count MBL with a median follow-up of 6.7 years were identified: 415 (63%) individuals were in Cohort A, 142 (22%) in Cohort B, and 100 in (15%) Cohort C. Compared to Cohort B, individuals in Cohort C were more likely to have unmutated IGHV, high expression of CD38, and higher beta-2 microglobulin level (Table 1). SLL patients were younger, had higher rate of unmutated IGHV, and had high expression of CD49d compared to MBL individuals in Cohort C (Table 1). There was suggestive evidence of a difference in TFT between Cohorts B and C (estimated 10-year treatment rate 28.3% vs. 41.6%; p=0.16; HR 1.62 95% CI 0.84-3.15). TFT was significantly shorter in SLL patients compared to those with Cohort C (estimated 10-year treatment rate 71% vs. 41.6%, respectively, P<0.001; Figure 1A). There was a trend toward shorter OS in Cohort C compared to Cohort B (estimated 10-year OS rate 53.7% vs. 64.2%, respectively, P=0.09; Figure 1B). In contrast, there was no difference in OS among MBL individuals in Cohort C compared to those with SLL (estimated 10-year OS rate 56.9% vs. 62.6%, P=0.72). Summary/Conclusion: Our findings demonstrate that individuals with MBL who have incidentally identified lymphadenopathy/splenomegaly at diagnosis are more likely to have an unfavorable risk profile, shorter TFT and OS compared to patients who did not have lymphadenopathy/splenomegaly at MBL diagnosis. Additionally, these individuals have a longer TFT but similar OS compared to SLL patients seen during the same time interval. If externally validated, these findings have significant implications for individuals with newly diagnosed MBL. Disclosures Ding: Merck: Research Funding; DTRM Biopharma: Research Funding. Kenderian:Novartis: Patents & Royalties, Research Funding; Tolero: Research Funding; Humanigen: Other: Scientific advisory board , Patents & Royalties, Research Funding; Lentigen: Research Funding; Morphosys: Research Funding; Kite/Gilead: Research Funding. Kay:MorphoSys: Other: Data Safety Monitoring Board; Infinity Pharmaceuticals: Other: DSMB; Celgene: Other: Data Safety Monitoring Board; Agios: Other: DSMB. Parikh:AstraZeneca: Honoraria, Research Funding; Janssen: Research Funding; AbbVie: Honoraria, Research Funding; Acerta Pharma: Research Funding; Ascentage Pharma: Research Funding; Genentech: Honoraria; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding.

Cancer cells have altered energy demand due to their increased proliferative capacity. In Waldenstrom Macroglobulinemia (WM), an indolent yet incurable B-cell lymphoma, the homeostasis of the bone marrow (BM) environment is disturbed due to the infiltration of the lymphoplamacytic cells that continuously produce monoclonal IgM. An alteration in energy demand could skew the balance of key proteins and metabolites towards a permissive niche for WM tumor cells growth, and unfavorably effect on the immune response against tumor cells. Therefore, the aim of our study was to identify how changes in certain metabolites and/or proteins could contribute to the pathobiology of WM and whether the cytokine and chemokine composition of the BM microenvironment play a role in such changes. WM patient's samples including BM plasma, peripheral blood serum and BM cells (n=101) as well as equivalent normal counterparts (n=86) were collected and used for metabolomics analysis. Comprehensive targeted metabolomics analysis was performed using Capillary Electrophoresis Time-of-Flight Mass spectrometry (CE-TOFMS), CE-triple quadrupole mass spectrometry (CE-qQqMS) and Liquid Chromatography (LC-TOFMS). Normal and WM peripheral blood serums samples were also used for untargeted proteomics analysis using a fully automated proteomic technology platform that includes an Agilent 1200 Series Quaternary HPLC system connected to a Q Exactive Plus mass spectrometer. Real-time PCR analysis was performed to detect the gene expression of the relevant metabolite transporters located on the cell membrane. BM cells from control and WM patients' samples were used for flow cytometry analysis. IHC was used to detect the proteins on the BM tissues. Principal Component Analysis (PCA) and Hierarchal Clustering Analysis (HCA) on both metabolomics and proteomics data identified two distinct clusters for disease and normal samples, indicating that there are differentially expressed proteins and metabolites in WM versus normal samples. Furthermore, pathway analysis showed that the majority of the altered metabolites were the members of the glutathione (GSH) metabolism pathway. This finding was further validated not only by data obtained from metabolomics analysis of BM cells and BM plasma, but also by proteomic data WM patients serum, implying that GSH metabolism is key to the biology of WM. Moreover, stimulation of WM cell lines by IL-6 and IL-21, cytokines involved in inducing WM cell proliferation and IgM secretion, resulted in increased gene expression of the transporters mediating uptake of metabolites required for GSH synthesis, including SLC7A11, 4F2HC and LAT1, indicating that cytokines in the WM BM could modulate GSH metabolism. In addition, IHC staining of the BM tissues as well as flow cytometry analysis of patients' lymphoplasmacytic cells identified glutathione peroxidase as one of the major proteins modulating GSH metabolism in WM. In summary, our data highlight a central role for GSH metabolism in WM disease biology and indicate that intervening with the metabolic processes could be a potential therapeutic strategy for patients with WM. Disclosures Ansell: AI Therapeutics: Other: research funding for clinical trials; Seattle Genetics: Other: research funding for clinical trials; Regeneron: Other: research funding for clinical trials; Affimed: Other: research funding for clinical trials; Bristol Myers Squibb: Other: research funding for clinical trials; Pfizer: Other: research funding for clinical trials; Merck: Other: research funding for clinical trials; Takeda: Other: research funding for clinical trials.

Introduction: Whereas morphological assessment and cytogenetics have been the cornerstone for the diagnosis of myelodysplastic syndrome (MDS), flow cytometry and mutational analyses are novel, evolving techniques. These diagnostic procedures play a role in identifying pre-MDS cases including idiopathic cytopenia of undetermined significance (ICUS) and clonal cytopenia of undetermined significance (CCUS). Aim: To assess flow cytometric findings in ICUS and CCUS and their correlation with progression to MDS or myeloid neoplasm (MN), concurrent genetic aberrations, and underlying pathological evolution. Methods: Patients (Pts) who had undergone evaluation to rule out MDS and had retrospectively undergone flow cytometry, cytogenetic, mutational and morphological assessment were included. Flow cytometry results were classified as normal (no abnormality or 1 equivocal result), abnormal (2 abnormalities or at least 1 abnormality and 1 equivocal result), or atypical (1 abnormality or 2-3 equivocal results). Next generation sequencing included mutation analysis of: ASXL1, BCOR, BRAF, CALR, CBL, CEBPA, CSF3R, DNMT3A, ETV6, EZH2, FLT3, GATA1, GATA2, IDH1, IDH2, JAK2, KIT, KRAS, MPL, MYD88, NOTCH1, NPM1, NRAS, PHF6, PTPN11, RUNX1, SETBP1, SF3B1, TERT, TET2, TP53, U2AF1, WT1, ZRSR2. Results: A) Characteristics: 229 pts were assessed (median age 60 and 69% were males). Based on pathologist interpretation, 87 (38%) pts met diagnostic criteria of MDS, 106 (46%) pts had normal/reactive bone marrow (BM), and 36 (16%) pts had BM findings suspicious for MDS. Median time to follow-up was 24 months for MDS pts, and 32 months for pts with cytopenias. B) Morphology: Pts who had a BM biopsy that was suspicious for MDS by pathologist interpretation were more likely to develop MDS compared to pts with normal/reactive pathology report with an incidence rate (IR) of 16% versus 0.9% (p= .0005). Pts who were suspicious for MDS based on the BM biopsy were more likely to have underlying mutations compared to pts with normal/reactive BM (IR 83% versus 69%, p= .2). C) Flow cytometry: In the non-MDS cohort, 96 (67%) pts had normal flow results, 29 (20%) pts had atypical flow, and 17 (12%) pts had abnormal flow results compared to 16 (18%) pts, 18 (21%) pts, and 53 (61%) pts in the MDS cohort, respectively. Of the pts who had normal flow result (n=112), 16 were MDS, 16 were suspicious for MDS/MN, and 80 were normal/reactive. Pts who had biopsy interpretation suspicious for MDS/MN were more likely to have abnormal flow cytometry compared to normal/reactive BM biopsy (IR 55% versus 24%, p=.0003). IR of MDS was 4% in normal flow, 3% in atypical flow, and 12% in abnormal flow (p= .5). Likewise, IR of MN was 4% in normal flow, 20% in atypical flow, and 30% in abnormal flow (p= .1). D) Genetics (cytogenetics and mutation profiling): Cytogenetics was normal in 123 (86%) pts and abnormal in 19 (13%) pts. Pts who had abnormal cytogenetics were more likely to develop MN (IR 57% versus 5%, p= .002). Out of the 50 non-MDS pts tested for NGS, 38 (76%) were positive for mutations. 15 (39%) pts had 1 mutation, 8 (21%) pts had 2, 3 (8%) pts had 3, 6 (16%) pts had 4, 2 (5%) pts had 5, 1 (3%) pt had 7, and 1 (3%) pt had 8 mutations (61% had >1 mutation). Pts with positive mutations were more likely to develop MDS during follow-up compared to pts who did not have mutations (IR 16% versus 0%, p=.06). Additionally, flow cytometric aberrations significantly correlate with the number of mutations (p= .03). MN correlated significantly with BCOR mutation and RUNX1 (IR 60% (p= .008) and 50% (p=.02), respectively. E) Interaction between variables: In pts with positive mutations, flow cytometry did not correlate with progression to MDS with an IR of 17% of MDS in normal flow, 11% in atypical flow, and 17% in abnormal flow, (p= .9). There was no impact on overall survival neither in pts with abnormal flow results vs normal nor in pts with positive mutations vs wild type. Conclusion: Conventional diagnostic techniques remain the cornerstone of MDS diagnosis with flow cytometry and NGS arising as novel techniques that correlate significantly with progression of pre-MDS pts to MDS. Flow had an impact on pathology final interpretation but did not add value in pt with a positive mutation analysis. Larger studies are needed to confirm our findings. Disclosures Patnaik: Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees. Al-Kali:Astex Pharmaceuticals, Inc.: Research Funding.