ALBERT

Description: Health professionals play an important role in educating the public about food safety risks. However, the ways this important group of educators remains up-to-date on these topics are not well defined. In this study, a national sample of dietitians employed in direct teaching of patients (n = 327) were recruited to complete a web-delivered survey designed to develop a model of factors that promote information processing and teaching in practice about food safety related to fresh vegetables. The resulting mental model demonstrates that dietitians teach fresh vegetable safety using systematic information processing to intellectually understand new information, but this is also associated with a gap in the dietitian’s knowledge of food safety. The juxtaposition of an information processing model with a behavioral model provides valuable new insights about how dietitians seek, acquire and translate/transfer important information to move patients toward a higher goal of food safety. The study also informs food safety educators as they formulate teaching strategies that are more effective than other approaches at promoting behavior change.

Description: Several types of tumor cells utilize the phosphatidylinositol 3 Kinase (PI3K)/AKT pathway to support their growth and survival, and blockade of this pathway has been shown to have an antiproliferative effect in these cells. The significance of this survival pathway in Hodgkin disease (HD), and in particular, the neoplastic Hodgkin and Reed-Sternberg (H/RS) of Hodgkin disease (HD) is unknown. We studied routinely processed tissue sections of 42 cases of primary HD (n=42, 35 classical HD and 7 nodular lymphocyte predominant) for the expression of the active phosphorylated form of AKT. We also studied AKT expression in a panel of 4 well-characterized HD-derived cell lines (HD-MyZ, HD-LM2, L-428, and KM-H2). 27 of 42 (64.3%) cases of primary HD sections expressed phospho AKT (23/35 classical and 4/7 nodular lymphocyte predominant). Cultured H/RS cells also showed constitutive phosphorylation of AKT on Ser473. CD30 ligand (CD30L), CD40L and RANKL, increased the phosphorylation of AKT and downstream molecules as early as 1 hour. The effect of 3 small molecule inhibitors of PI3K/AKT pathway (PI3K inhibitor LY294002; AKT inhibitors QLT0394 and QLT0395 from QLT Inc, Vancouver, Canada) on cell proliferation and survival of cultured H/RS cells was examined by the MTS assay. The PI3K inhibitor LY294002 showed antiproliferative activity in a time and dose dependent manner in all cell lines tested. This antiproliferative effect was primarily due to cell cycle arrest in G0/G1 phase, as determined by propidium iodide staining, and to a less extent due to induction of apoptosis, as determined by the Annexin-V binding assay. However, when the AKT inhibitors QLT0394 and QLT0395 were used, they primarily induced apoptosis even at nanomolar doses. Apoptosis was mediated by caspase 8 activation as determined by western blot and was partially reversed by the pancaspase inhibitor ZVAD-FMK. Both AKT inhibitors alteredf AKT phosphorylation within 24 hours, and reduced phosphorylation of downstream molecules, such as 4E-BP1. Moreover, the AKT inhibitor QLT0395 showed downregulation of MDM2 and cyclin D1 within 24 hours, and increased ERK1/2 phosphorylation with subsequent decrease of total ERK1/2 levels at the same time frame, but had no effect in BCL-2, cFLIP or Bax. Importantly, both AKT inhibitors (QLT0394 and QLT0395) maintained their killing activity even in the presene of CD30L, CD40L, and RANKL. These preclinical data suggest that small molecules hitting different targets within PI3K/AKT pathway may have different effects on proliferation and apoptosis and that blockade of this pathway may be of therapeutic value in the treatment of patients with HD.

Description: Introduction: PTL is a rare extranodal lymphoma characterized by a continuous pattern of systemic relapses in spite of doxorubicin-based therapy. There are also frequent relapses in the contralateral testis and the central nervous system (CNS). Therefore we decided to determine whether prophylaxis of the CNS and the contralateral testis would modify the pattern of relapse of PTL. Patients and methods: From 1993 to February 2002 in IELSG member institutions patients with PTL (presenting with testicular enlargement) were managed with doxorubicin-based therapy according to institutional policy, with the addition of four doses of IT methotrexate (15 mg) during the first 6 weeks. At the end of chemotherapy scrotal RT (30 Gy) was given to all patients and 30–36 Gy nodal RT to those with stage II disease. Rituximab was not used in this cohort because it was not yet generally available for patients with diffuse large B-cell lymphoma. Results: There were twenty seven patients; two refused chemotherapy and/or RT/IT therapy and one RT, leaving 24 eligible for analysis. Median age was 60 years (range 31–80). Ann Arbor stage was I in 17, II in four, and four in three patients. B-symptoms were present in one patient. The right testis was involved in 13 and the left in 11 patients. Serum LDH was elevated in four of 21 with available values. No patient had CNS involvement at diagnosis. Pathology was diffuse large B-cell lymphoma in all patients. Treatment was CHOP in 21, alternating triple therapy in two, and hyper-CVAD in one patient. Three patients died during therapy: one from neutropenic sepsis and two from toxicity of systemic methotrexate. The remaining 21 patients achieved a complete remission. With a median follow-up of 57 months (range 8–123) for survivors, seven patients have relapsed, three in the CNS. There were no testicular relapses. Eight patients have died: three from toxicity during induction, three from relapsed lymphoma, one from gastric carcinoma and one from sudden death, both in complete remission. At 5 years the projected progression-free survival was 78%, and survival 66%, but both without an apparent plateau. Freedom from progression in the CNS was 84%, with a plateau. Conclusions: Patients with PTL treated prospectively with doxorubicin-based therapy before the availability of rituximab exhibit the continuous pattern of relapse previously reported. Scrotal RT seems eliminate testicular relapses, and prophylactic IT methotrexate seems to reduce, but not eliminated CNS relapses. These need to be confirmed by longer follow-up. The ongoing IELSG-10 should determine whether the addition of rituximab alters the continuous pattern of systemic relapses. Future studies should determine the molecular basis of the continuous relapses and minimize both systemic and CNS relapses.

Description: Background Isocitrate dehydrogenase 1 (IDH1) and IDH2 mutations are important prognostic biomarkers in acute myeloid leukemia (AML). Although the clinicopathologic correlates of IDH mutations have been extensively studied, the distribution of abnormal myeloid cells carrying these mutations has not been studied. Specific localization of cells carrying IDH mutations will be useful in further understanding the pathophysiology and post-treatment biology of IDH mutant cases of AML. This characterization is becoming particularly relevant for identification of minimal residual disease, especially for patients treated with novel IDHinhibitors. In this study, we characterized IDH1 p.R132H clones in bone marrow specimens involved by AML using a mutation specific antibody. Materials and Methods Bone marrow tissue sections (biopsy or clot specimens) from 32 AML cases with IDH1 p.R132H mutation were stained with IDH1 p.R132H-mutation specific antibody. These cases include 20 de novoAML and 12 cases of AML with myelodysplasia-related changes (AML-MRC). We also included 10 AML cases with wild-type IDH1 as a control. After confirmation of the positive IDH1 immunohistochemical (IHC) signal in the primary specimens, follow up bone marrow specimens (n=67) including (a) persistent disease, (b) minimal residual disease by flow cytometry, (3) complete remission by morphology and flow cytometry, but, positive for mutation by PCR, as well as (4) relapsed cases after complete remission were included in the study (in progress). We also included pre- and post-treatment (unresponsive with increasing blast counts, stable disease, persistent disease with decreasing blast counts, complete remission, and relapse) bone marrow specimens (n=72) from 16 patients treated with IDH inhibitors (in progress). Results All the IDH1 wild type AML cases were negative for IDH1 IHC stain showing 100% specificity. Positive signal was detected in all de novo AML and AML-MRC (allelic frequency ranges from 1.8% to 47% by PCR) except one AML case with 8.9% allele burden which was a limited sample; overall sensitivity was 96%. The IHC signal was detected in the cytoplasm of myelomonocytic cells, their precursors, and megakaryocytes. Erythroid precursors, lymphoid cells, endothelial cells, and osteoblasts were consistently negative. The signal intensity ranged from weak (n=10) to moderate (n=9), to strong (n=13). The positive cells predominantly showed an interstitial distribution in the bone marrow. In the de novo AML group, only the immature cells were positive in 100% of pre-treatment AML cases. However, both mature and immature cells were positive in 7/13 (54%) post-treatment AML cases (6 cases treated with hypomethylating agents). One case was transformed from MPN which also showed positivity in mature and immature cells. In two cases with complete morphologic remission and one case with minimal residual disease detected by flow cytometry, IHC signal was detected in both mature and immature cells; both patients relapsed in 8 and 11 months. In the AML-MRC group, both immature and mature cells were positive in 11/12 (92%) cases of which 2 were not previously treated indicating the possibility that IDH1 mutation is an early event. Since the remaining 9 patients were treated with hypomethylating agents, the positivity of both mature and immature cells as a result of maturation effect versus an early event cannot be assessed. Additional studies for follow-up AML cases, including cases on an IDH inhibitor clinical trial are in progress. Conclusions Our preliminary data indicate that IDH1 IHC is a highly specific and sensitive tool to detect IDH1 R132H mutated cases and can be used as a primary method to localize the population of mutation-bearing cells in the bone marrow. IHC also allows determination of whether the IDH1 mutation in the post-treatment setting is arising from immature or mature cells. IHC provides an opportunity to understand the difference between these two populations and, based on characterization of cell type and distribution, may be helpful to predict whether the risk of relapse is high. Disclosures DiNardo: Agios: Other: advisory board, Research Funding; Novartis: Other: advisory board, Research Funding; Daiichi Sankyo: Other: advisory board, Research Funding; Celgene: Research Funding; Abbvie: Research Funding.

Description: We have developed a stromal-based in vitro culture system that facilitates ex vivo expansion of transplantable CD34+thy-1+ cells using long-term hematopoietic reconstitution in severe combined immunodeficient-human (SCID-hu) mice as an in vivo assay for transplantable human hematopoietic stem cells (HSCs). The addition of leukemia inhibitory factor (LIF) to purified CD34+ thy-1+ cells on AC6.21 stroma, a murine bone marrow–derived stromal cell line, caused expansion of cells with CD34+ thy-1+ phenotype. Addition of other cytokines, including interleukin-3 (IL-3), IL-6, granulocyte-macrophage colony-stimulating factor, and stem cell factor, to LIF in the cultures caused a 150-fold expansion of cells retaining the CD34+ thy-1+ phenotype. The ex vivo–expanded CD34+ thy-1+ cells gave rise to multilineage differentiation, including myeloid, T, and B cells, when transplanted into SCID-hu mice. Both murine LIF (cannot bind to human LIF receptor) and human LIF caused expansion of human CD34+ thy-1+ cells in vitro, suggesting action through the murine stroma. Furthermore, another human HSC candidate, CD34+ CD38− cells, shows a similar pattern of proliferative response. This suggests thatex vivo expansion of transplantable human stem cells under this in vitro culture system is a general phenomenon and not just specific for CD34+ thy-1+ cells.

Description: Introduction: Acalabrutinib and ibrutinib are Bruton's tyrosine kinase (BTK) inhibitors, which have revolutionized the treatment of B-cell lymphoma/leukemias such as mantle cell lymphoma (MCL) and chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) in frontline or relapsed refractory setting. Outcomes of patients with MCL and/or CLL who progressed on ibrutinib therapy are poor. Few reports have shown that ibrutinib resistant patients develop specific BTK mutations which confer resistance. We present the clinical and molecular data on patients with follicular lymphoma (FL) who have progressed on either ibrutinib based regimen or on acalabrutinib. Methods: We selected FL patients enrolled in clinical studies at our institution between 2013 and 2015 who discontinued acalabrutinib or ibrutinib for various reasons. Survival post ibrutinib discontinuation was calculated. We performed whole-exome sequencing on pre and post treatment (paired) samples of 3 FL patients (progressed on ibrutinib) using DNA extracted from formalin fixed paraffin embedded tissue samples. Library preparation and capture were performed using Agilent's SureSelect XT Human All Exon V4. Following capture the enriched libraries were pooled and sequenced in two Illumina HiSeq4000 lanes using a 75nt paired-end configuration. Results: Twenty of 55 patients discontinued treatment with BTK inhibitors; 5 came off from single agent acalabrutinib in relapsed refractory FL (RR-FL) while 15 came off from ibrutinib based regimen (4 as a single agent in RR-FL while 11 were treated with ibrutinib and rituximab in the frontline setting). These patients discontinued BTK inhibitor treatment, after a median of 2.8 months (range 0.9-22 months). Patients who received frontline ibrutinib (n=11), discontinued at a median of 2.8 months (0.9-22 months); previously treated patients discontinued BTK inhibitors at a median of 2.8 months (1.4-7.6 months). The reasons for discontinuation were as follows - 14 disease progression (3 on acalabrutinib and 11 on ibrutinib; 1 of them developed classical Hodgkin lymphoma), 2 non-responders (1 acalabrutinib and 1 ibrutinib) and 4 for other reasons (1 due to fatigue on acalabrutinib, 3 due to ibrutinib - 1 elevated liver enzymes, 1 asymptomatic atrial fibrillation another lost to follow up). Pre-treatment characteristics of the 16 patients who either progressed or were nonresponders to BTK inhibitors were - median age 59 years (range 41-75), 69% had stage 4 disease, 31% had FLIPI score of 3 or 4 and median LDH levels were 457 IU/L (range 290-754). Median follow up after discontinuation for all patients was 14.4 months (0-32 months). Eighteen patients (90%) were alive at the time of last follow up and 2 (10%) have died. Median survival (figure 1) of patients according to the causes of discontinuation was: not reached for both group of patients, those with other causes and those in progressive disease and/or non-responders. All 16 patients who progressed or not responded were treated with additional therapies. At the time of last follow up, 10/16 (62%) were responding to subsequent therapy - 5/10 (62%) achieved CR on lenalidomide-based therapies, 4/10 (40%), responded to chemoimmunotherapy and 1 patient responded to venetoclax-bendamustine and rituximab with CR. None were treated with PI3K inhibitors. WES studies showed EZH2 Y646 mutations in 2 of 3 FL patients in both pre and post treatment samples. Comparison of pre and post treatment samples on 3 patients showed multiple non-recurrent variants (2-12 per patient) in several genes in the post-treatment samples. These included DNMT3A R882H, NOTCH2 A3D and MLL E765G, all of which were noted in the post treatment sample of the patient that progressed to CHL. None of the other detected variants have been implicated in cancer. Of note, none of the samples showed BTK mutations despite adequate coverage. Conclusions: In contrast to other B-cell malignancies such as CLL and MCL, we observed that almost all FL patients who progressed on BTK inhibitors were salvageable by alternate therapies. Absence of BTK gene mutations in paired samples of 3 patients who progressed on ibrutinib suggests alternate mechanisms of drug resistance. Figure 1 Outcomes of patients with follicular lymphoma after discontinuing acalabrutinib and/or ibrutinib based regimen according to the cause of discontinuation Figure 1. Outcomes of patients with follicular lymphoma after discontinuing acalabrutinib and/or ibrutinib based regimen according to the cause of discontinuation Disclosures Wang: Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno Therapeutics: Research Funding; Onyx: Research Funding; Kite Pharma: Research Funding; Asana BioSciences: Research Funding; Celgene: Research Funding; Pharmacyclics: Research Funding; BeiGene: Research Funding. Fayad:Seattle Genetics: Consultancy, Research Funding. Oki:Novartis: Research Funding. Westin:Spectrum: Membership on an entity's Board of Directors or advisory committees; Chugai: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; ProNAi: Membership on an entity's Board of Directors or advisory committees. Fowler:Gilead: Research Funding; Jannsen: Consultancy, Research Funding; Infinity: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; TG Therapeutics: Consultancy.

Description: Background: Dendritic cell sarcomas (DCS) mainly include follicular dendritic cell sarcoma (FDCS) and interdigitating cell sarcoma (IDCS). DCS are difficult to diagnose. There is no standard of care and research on DCS is sparse due to its rarity. We present our data on the clinico-pathological characteristics, treatments and survival of patients (pts) with DCS. Methods: This is a retrospective analysis of 64 pts with confirmed diagnosis of DCS (FDCS n=58; IDCS n=6) evaluated at MDACC (1995 and 2015). Following IRB approval, pt data were collected on the clinico-pathological features, treatments and clinical outcomes. Histopath. and immunohistochemistry (IHC) data were collected. Results: Median age at diagnosis was 51 yrs (range 16-77 yrs). 35 pts were female and 29 were male. 51% pts presented with localized disease and 49% with systemic involvement. Overall, 13 pts had nodal involvement, 21 had extra nodal disease and 21 had both. The abdomen was involved in 33/58 pts with liver (9/33) followed by spleen (5/33) as the most common extranodal sites of disease. 12 pts (19%) had pre-existing or subsequent autoimmune disease (3 had pemphigus, 3 thyroid disease and 2 had myasthenia gravis and 4 had other). 6 pts with FDCS had Castleman's disease (hyaline vascular). IHC markers in FDCS were clusterin, EGFR, vimentin, CD21, CD35 and CD23 in 90%, 81%, 87%, 81%, 80% and 64% samples. Ki-67 was 〉 25% in 44% pts. S-100 and CD68 were expressed in 11% and 28% respectively. For IDCS, markers included S-100 and CD68 in 100% and 83% pts. Electron microscopy was performed in 3 pts and revealed neoplastic cells with well-formed junctions and very prominent desmosomes. Molecular studies in 2 pts were negative for BRAF mutations. One pt had TP53 exon 8 and another had PTEN mutation. 50 pts had treatment information available, and 45 had response data. Initial treatments included surgery followed by adjuvant chemotherapy with/without radiation in (21/50), chemotherapy alone in (7/50), radiation with/without chemotherapy in 6 pts and surgery with radiation in 6 pts. The overall response rate (ORR) to initial therapy was 78% (35/45) - FDCS 78%; 32/41 and IDCS 3/4; 75%. Complete response (CR) was observed in 40% (18/45) - FDCS 41%; 17/41 and IDCS 1/4; 25%. Partial response rate (PR) was 38% (17/45) - FDCS 37%; 15/41 and IDCS 2/4; 50%. In pts who received chemotherapy, gemcitabine with taxanes (Gem-Tax) (n=12) and CHOP based regimens (n=12) were most commonly used and were associated with ORR of 83% (10/12) and 75% (9/12) respectively. The CR rate following chemotherapy (alone or following local therapy) with both Gem-Tax and CHOP was 42%. Among pts who received Gem-Tax with/without other modalities - 42% (5/12) were CR and 41% were PR (5/12) and 2 pts were non responders. For the 10 responders, 6 pts had surgery prior to chemotherapy, 3 had radiotherapy and 1 had chemotherapy alone. For pts who received CHOP based regimens with/without other modalities - 42% (5/12) were CR and 33% were PR (4/12) and 3 pts did not respond. For the 9 responders, 6 pts had surgery prior to chemotherapy, and 3 had radiotherapy. Eight pts were treated with miscellaneous regimen as first line, Ifos. with Adria. (3) and one each with R-COP, R-CHOP, lenalidomide with prednisone, imatinib and ICE with surgery or radiotherapy. Overall, 27 pts were alive (23 FDCS and 4 had IDCS) and 33 pts died (31 FDCS and 2 had IDCS) at the time of last follow up - (median 45 months; range 5-129 months). Overall, median progression free and overall survival was 33 and 50 months respectively. Furthermore, among the FDCS, survival outcomes were significantly shorter in pts with extra nodal disease as compared to nodal disease alone (Figure 1A-B- p=0.018 for PFS and p=0.030 for OS). Conclusions: We present the largest single center report of pts with DCS. We observed marginally higher responses with gemcitabine/docetaxel combinations compared to CHOP based therapy, but limited numbers make comparisons difficult and prospective studies are needed to define optimal treatment in DCS. Combined modality treatment appears to result in improved outcomes. Moreover, we have shown that pts with nodal disease alone at the time of initial presentation have significantly longer survival. We are studying the genomic profile in DCS and identify potential targets for therapy. Figure 1. Progression free and overall survival in patients FDCS Figure 1. Progression free and overall survival in patients FDCS Disclosures Wang: Celgene: Research Funding. Orlowski:Bristol-Myers Squibb: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; BioTheryX, Inc.: Membership on an entity's Board of Directors or advisory committees; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Forma Therapeutics: Consultancy; Spectrum Pharmaceuticals: Research Funding; Onyx Pharmaceuticals: Consultancy, Research Funding; Acetylon: Membership on an entity's Board of Directors or advisory committees; Genentech: Consultancy; Millennium Pharmaceuticals: Consultancy, Research Funding; Array BioPharma: Consultancy, Research Funding.

Description: Introduction: Salvage immunochemotherapy (IC), followed by high-dose chemotherapy with autologous stem cell transplantation if chemosensitive is standard-of-care second-line (2L) therapy (tx) for fit patients (pts) with relapsed/refractory (R/R) diffuse large B cell lymphoma (DLBCL) when treated with first-line (1L) R-CHOP as shown in the CORAL study. Outcomes following receipt of salvage IC in the 2L setting for pts with DLBCL or high grade B cell lymphoma/B cell lymphoma unclassifiable (HGBL/BCLU) receiving intensive 1L tx remain unknown, and may be worse than those reported in CORAL given prior exposure to higher-dose IC in 1L setting. Here we report the results of a multicenter retrospective analysis of R/R DLBCL and HGBL/BCLU pts treated with intensive 1L tx who receive standard salvage 2L tx. Methods: Inclusion criteria were histologic diagnosis of DLBCL or HGBL/BCLU, R/R disease following 1L tx with R-EPOCH, R-HyperCVAD or R-CODOX-M/IVAC and receipt of 2L tx with R-ICE, R-DHAP, R-DHAC, R-ESHAP or R-GDP. Exclusion criteria were HIV positivity, post-transplant lymphoproliferative disorder, prior chronic lymphocytic leukemia and inadequate data. Therapy was given at the discretion of the treating physician. Progression free survival (PFS) was defined as the interval between time of first relapse or primary refractory disease and disease progression, change in therapy if no disease response or last follow-up in remission, and overall survival (OS) between time of first relapse or primary refractory disease and death or last follow-up while alive. Pts were treated from 2007-2017 and data were censored on 10/15/17. Results: A total of 195 pts treated at 20 US and Canadian academic medical centers were included. Clinicopathologic characteristics at time of R/R disease were 39% age 〉60 years, 62% male, 77% stage III-IV, 72% elevated LDH, 24% bone marrow (BM) involvement, 28% B symptoms present, 44% extranodal (EN) disease at 〉1 site, 19% ECOG performance status (PS) 〉1, 49% with International Prognostic Index score (IPI) ≥3, 46% HGBL/BCLU histology, 49% Ki67 ≥90%, and 61% germinal center (GCB) cell of origin (COO) by Hans algorithm. Of pts with available fluorescence in situ hybridization (FISH) data, 51%, 45% and 30% demonstrated MYC, BCL2 and BCL6 rearrangements (-R), respectively, and 37% were double hit lymphoma (DHL). Tx received in 1L were R-EPOCH in 82%, R-HyperCVAD in 16% and R-CODOX-M/IVAC in 2% of pts. R-ICE was received by 64% and other platinum-containing regimens by 36% as 2L tx. Most (86%) pts relapsed within 12 months (mo) of completion of 1L tx (early) and 58% of pts had primary refractory disease. For all pts, the median length of follow-up was 25.0 mo with a median PFS of 3.0 mo and median OS of 8.0 mo. Overall response rate to 2L tx among all pts was 44% (23% complete response [CR] and 21% partial response [PR]), and 48% with progressive disease. Pts achieving CR had significantly longer median PFS (32.0 vs 4.0 mo, p = 0.0001) and OS (not reached vs 13.0 mo, p = 0.0004) as compared to pts achieving PR. In pts who achieved CR or PR following 2L tx, 64% received consolidative transplant (42 autologous and 13 allogeneic) and achieved a median PFS and OS of 18.3 mo and 62.0 mo, respectively. As compared to pts relapsing ≥12 mo after completion of 1L tx (late), pts relapsing early were less likely to achieve CR (17% vs. 61%, p=0.0001) and experienced significantly shorter median PFS (2.8 vs. 23.0 mo, p1 and Ki67 ≥90%, but not BCL2-R, demonstrated a statistically significant increased HR for death. Multivariate analysis demonstrated only early relapse to have a statistically significant increased HR for progression (HR 2.47, p=0.024) and death (HR 5.90, p=0.001). Conclusion: Relapse

Description: We have developed a stromal-based in vitro culture system that facilitates ex vivo expansion of transplantable CD34+thy-1+ cells using long-term hematopoietic reconstitution in severe combined immunodeficient-human (SCID-hu) mice as an in vivo assay for transplantable human hematopoietic stem cells (HSCs). The addition of leukemia inhibitory factor (LIF) to purified CD34+ thy-1+ cells on AC6.21 stroma, a murine bone marrow–derived stromal cell line, caused expansion of cells with CD34+ thy-1+ phenotype. Addition of other cytokines, including interleukin-3 (IL-3), IL-6, granulocyte-macrophage colony-stimulating factor, and stem cell factor, to LIF in the cultures caused a 150-fold expansion of cells retaining the CD34+ thy-1+ phenotype. The ex vivo–expanded CD34+ thy-1+ cells gave rise to multilineage differentiation, including myeloid, T, and B cells, when transplanted into SCID-hu mice. Both murine LIF (cannot bind to human LIF receptor) and human LIF caused expansion of human CD34+ thy-1+ cells in vitro, suggesting action through the murine stroma. Furthermore, another human HSC candidate, CD34+ CD38− cells, shows a similar pattern of proliferative response. This suggests thatex vivo expansion of transplantable human stem cells under this in vitro culture system is a general phenomenon and not just specific for CD34+ thy-1+ cells.

Description: The development of culture systems that facilitate ex vivo maintenance and expansion of transplantable hematopoietic stem cells (HSCs) is vital to stem cell research. Establishment of such culture systems will have significant impact on ex vivo manipulation and expansion of transplantable stem cells in clinical applications such as gene therapy, tumor cell purging, and stem cell transplantation. We have recently developed a stromal-based culture system that facilitates ex vivo expansion of transplantable human HSCs. In this stromal-based culture system, 2 major contributors to the ex vivo stem cell expansion are the addition of leukemia inhibitory factor (LIF) and the AC6.21 stromal cells. Because the action of LIF is indirect and mediated by stromal cells, we hypothesized that LIF binds to the LIF receptor on AC6.21 stromal cells, leading to up-regulated production of stem cell expansion promoting factor (SCEPF) and/or down-regulated production of stem cell expansion inhibitory factor (SCEIF). Here we demonstrate a secreted SCEPF activity in the conditioned media of LIF-treated AC6.21 stromal cell cultures (SCM-LIF). The magnitude of ex vivo stem cell expansion depends on the concentration of the secreted SCEPF activity in the SCM-LIF. Furthermore, we have ruled out the contribution of 6 known early-acting cytokines, including interleukin-3, interleukin-6, granulocyte macrophage colony-stimulating factor, stem cell factor, flt3 ligand, and thrombopoietin, to this SCEPF activity. Although further studies are required to characterize this secreted SCEPF activity and to determine whether this secreted SCEPF activity is mediated by a single factor or by multiple growth factors, our results demonstrate that stromal cells are not required for this secreted SCEPF activity to facilitate ex vivo stem cell expansion.