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Wei Zhang,1 Kimberly R Jordan,2 Brian Schulte,3 Enkhtsetseg Purev1 1Division of Hematology, Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, USA; 2Division of Immunology, Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, USA; 3Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, USA Background: Chimeric antigen receptor (CAR) T-cell therapy is highly effective for treating acute lymphoblastic leukemia and non-Hodgkin’s lymphoma with high rate complete responses. However, the broad clinical application of CAR T-cell therapy has been challenging, largely due to the lack of widespread ability to produce and high cost of CAR T-cell products using traditional methods of production. Automated cell processing in a closed system has emerged as a potential method to increase the feasibility of producing CAR T cells locally at academic centers due to its minimal reliance on experienced labor, thereby making the process less expensive and more consistent than traditional methods of production. Method: In this study, we describe the successful production of clinical grade CD19 CAR T cells using the Miltenyi CliniMACS Prodigy Automated Cell Processor at University of Colorado Anschutz Medical Campus in a rapid manner with a high frequent CD19 CAR expression. Results: The final CAR T-cell product is highly active, low in immune suppression, and absent in exhaustion. Full panel cytokine assays also showed elevated production of Th1 cytokines upon IL-2 stimulation when specifically killing CD19+ target cells.Conclusion: These results demonstrate the feasibility of producing CAR T cells locally in a university hospital setting using automated cell processor for future clinical applications. Keywords: automated decentralized cell production, CD19, chimeric antigen receptor, immunophenotype, activation status, cytokine panel

Background: Chimeric antigen receptor (CAR) T-cell therapy has improved the historically poor outcomes for relapsed and refractory (R/R) large B-cell non-Hodgkin's lymphoma (LBCL). However, nearly 60% of patients will either fail to respond or relapse after CAR T-cell therapy. Currently, PET/CT scans are used to assess response. Cell-free circulating tumor DNA (ctDNA) is released by tumor cells into the peripheral blood and can be measured for minimal residual disease (MRD) assessment., Methods: In this retrospective, IRB approved pilot study, archived lymphoma tissue and ctDNA from peripheral blood samples on day 0, 14, 28, 56, 90, 180, and 365 after CAR T-cell infusion from 10 patients with R/R NHL were collected for next-generation sequencing (NGS) of clonal variable-diversity-joining (VDJ) rearrangements (Adaptive biotechnologies [Seattle, WA]). Response was assessed by PET/CT on days 90 and 365 and graded according to the Lugano 2014 criteria. The primary endpoint was to determine the feasibility of detecting ctDNA to monitor disease response after anti-CD19 CAR T-cell therapy. The secondary endpoint was to compare the sensitivity/specificity of MRD assessment from ctDNA to PET/CT imaging., Results: Nine out of 10 patients with a trackable sequence [median age 69 (range: 56-76); 55.6% male; median LDH 224], were included in this study. Each received tisagenlecleucel (tisa-cel) CAR T-cell therapy after median 2 prior treatments (range: 2-4). 7/9 patients had R/R diffuse large B-cell lymphoma (DLBCL), and 2/9 had transformed follicular lymphoma. At a median follow up of 12.7 months (range: 1.5-30 months), 4 patients were alive. By day 90, 3 patients (33.3%) achieved a radiographic complete response (CR) whilst 6 patients (66.6%) had progressive disease (PD). Detectable MRD on day 14 or day 28 had 83% sensitivity and 100% specificity for radiographic progression at any time before 1 year. For patients with PD, the median (interquartile range) MRD at day 0, 14, and 28 were 17.31 (1.01, 96.84), 9.12 (0.30, 18.8), and 23.77 (8.01, 137.53) copies per milliliter (mL), respectively. For patients with detectable MRD at day 28, mOS and mPFS were 6.7 and 1.3 months, respectively., Conclusion: Monitoring MRD was a sensitive and specific method to detect poor response to tisa-cel. Additional studies evaluating MRD more frequently and with different products are warranted., Competing Interests: Disclosure MK receives research support from Novartis and performs consultant work for Adaptive Biotechnologies. AJ is employed by and is a shareholder of Adaptive Biotechnologies., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Despite the concurrent use of haploidentical cord (HCT) and dual cord (DCT) stem cell transplant approaches for over a decade, there have been few comparisons of their outcomes. Our objective in this study is to assess for differences in the outcomes and adverse effects associated with HCTs versus DCTs. Here we report a retrospective analysis of HCTs and DCTs at our institution. From October 2012 to October 2022, 70 HCT and 133 DCT transplants were performed following 50 mg/kg of IV cyclophosphamide, 150 mg/m 2 of IV fludarabine, 10 mg/kg of IV thiotepa, and 4 Gy total body irradiation conditioning. With a median follow-up of 3.6 years among survivors, there was no difference in overall survival (OS) (3 years OS 65% DCT versus 63% HCT, P = 1) or relapse-free survival (3 years RFS 62% DCT versus 64% HCT, P = .97) for all patients. Time to neutrophil recovery was faster in HCT recipients (median 17 versus 22 days, P = .021), with no difference in platelet recovery to 20,000/μL (P = .12). Median hospitalization for HCT recipients was 20 days versus 24 days for DCT recipients (P < .0001). Engraftment syndrome treated with steroids occurred in 47/133 (35%) DCT recipients versus 42/70 (60%) HCT recipients (odds ratios 0.37, P value=.001). There was a significant increase in grade 3 to 4 acute graft-versus-host disease (aGVHD) in haplo-cord recipients (P = .007), but no difference in grade 2 to 4 aGVHD (P = .11), all chronic GVHD (cGVHD) (P = .9), or moderate-severe cGVHD (P = .3). Our outcomes demonstrate faster engraftment and shorter hospitalization in HCTs relative to DCTs, but more engraftment syndrome and higher grade 3 to 4 aGVHD. When both are options, these factors should guide the choice between HCTs and DCTs., (Copyright © 2024. Published by Elsevier Inc.)

Abstract: Lisocabtagene maraleucel (liso-cel) demonstrated significant efficacy with a manageable safety profile as third-line or later treatment for patients with relapsed or refractory (R/R) large B-cell lymphoma (LBCL) in the TRANSCEND NHL 001 study. Primary end points were adverse events (AEs), dose-limiting toxicities, and objective response rate (ORR) per independent review committee. Key secondary end points were complete response (CR) rate, duration of response (DOR), progression-free survival (PFS), and overall survival (OS). After 2-year follow-up, patients could enroll in a separate study assessing long-term (≤15 years) safety and OS. Liso-cel-treated patients (N = 270) had a median age of 63 years (range, 18-86 years) and a median of 3 prior lines (range, 1-8) of systemic therapy, and 181 of them (67%) had chemotherapy-refractory LBCL. Median follow-up was 19.9 months. In efficacy-evaluable patients (N = 257), the ORR was 73% and CR rate was 53%. The median (95% confidence interval) DOR, PFS, and OS were 23.1 (8.6 to not reached), 6.8 (3.3-12.7), and 27.3 months (16.2-45.6), respectively. Estimated 2-year DOR, PFS, and OS rates were 49.5%, 40.6%, and 50.5%, respectively. In the 90-day treatment-emergent period (N = 270), grade 3 to 4 cytokine release syndrome and neurological events occurred in 2% and 10% of patients, respectively. The most common grade ≥3 AEs in treatment-emergent and posttreatment-emergent periods, respectively, were neutropenia (60% and 7%) and anemia (37% and 6%). Liso-cel demonstrated durable remissions and a manageable safety profile with no new safety signals during the 2-year follow-up in patients with R/R LBCL. These trials were registered at www.ClinicalTrials.gov as #NCT02631044 and #NCT03435796., (© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Lung organoids display a tissue-specific functional phenomenon and mimic the features of the original organ. They can reflect the properties of the cells, such as morphology, polarity, proliferation rate, gene expression, and genomic profile. Alveolar type 2 (AT2) cells have a stem cell potential in the adult lung. They produce and secrete pulmonary surfactant and proliferate to restore the epithelium after damage. Therefore, AT2 cells are used to generate alveolar organoids and can recapitulate distal lung structures. Also, AT2 cells in human-induced pluripotent stem cell (iPSC)-derived alveolospheres express surfactant proteins and other factors, indicating their application as suitable models for studying cell-cell interactions. Recently, they have been utilized to define mechanisms of disease development, such as COVID-19, lung cancer, idiopathic pulmonary fibrosis, and chronic obstructive pulmonary disease. In this review, we show lung organoid applications in various pulmonary diseases, drug screening, and personalized medicine. In addition, stem cell-based therapeutics and approaches relevant to lung repair were highlighted. We also described the signaling pathways and epigenetic regulation of lung regeneration. It is critical to identify novel regulators of alveolar organoid generations to promote lung repair in pulmonary diseases.

Up to 40% of patients with diffuse large B-cell lymphoma (DLBCL) are refractory to or relapse after first-line therapy, highlighting the need for better treatments. Mosunetuzumab is a CD20 × CD3 bispecific antibody that engages and redirects T cells to eliminate malignant B cells. In this phase 2, open-label study (NCT03677141), 40 patients (52.5% with international prognostic index ≥3) with previously untreated DLBCL initiated 6 cycles of IV mosunetuzumab with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) chemotherapy. Mosunetuzumab was administered in cycle 1 as step-up doses to mitigate cytokine release syndrome [CRS], and a dose of 30 mg was given on day 1 of cycles 2-6. Efficacy end points included objective and complete response rates, as determined by the investigator, via positron emission tomography-computed tomography, using Lugano 2014 criteria (87.5% and 85.0%, respectively). At a median follow-up of 32.0 months, the estimated 2-year progression-free survival and event-free survival rates were 65.4% (95% confidence interval [CI], 49.5-81.4) and 60.4% (95% CI, 44.7-76.1), respectively. CRS occurred in 60.0% of patients; all events were grade 1 (45.0%) or grade 2 (15.0%) and occurred primarily in cycle 1. Mosunetuzumab-related grade ≥3 neurologic adverse events (AEs) potentially consistent with immune effector cell-associated neurotoxicity syndrome occurred in 1 patient (2.5%). Grade 5 AEs were reported in 2 patients. Neutropenia occurred in 70.0% of patients, mostly during cycle 1 and was of short duration. These findings demonstrate promising activity and a manageable safety profile for mosunetuzumab-CHOP and warrant further investigation of mosunetuzumab in first-line combination regimens for DLBCL., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Over the past 15 years, and despite many difficulties, significant progress has been made to advance child and adolescent tuberculosis (TB) care. Despite increasing availability of safe and effective treatment and prevention options, TB remains a global health priority as a major cause of child and adolescent morbidity and mortality-over one and a half million children and adolescents develop TB each year. A history of the global public health perspective on child and adolescent TB is followed by 12 narratives detailing challenges and progress in 19 TB endemic low and middle-income countries. Overarching challenges include: under-detection and under-reporting of child and adolescent TB; poor implementation and reporting of contact investigation and TB preventive treatment services; the need for health systems strengthening to deliver effective, decentralized services; and lack of integration between TB programs and child health services. The COVID-19 pandemic has had a significant negative impact on case detection and treatment outcomes. Child and adolescent TB working groups can address country-specific challenges to close the policy-practice gaps by developing and supporting decentral ized models of care, strengthening clinical and laboratory diagnosis, including of multidrug-resistant TB, providing recommended options for treatment of disease and infection, and forging strong collaborations across relevant health sectors.

Cell based therapies including chimeric antigen receptor (CAR) T cells are promising for treating leukemias and solid cancers. At the same time, there is interest in enhancing the functionality of these cells via surface decoration with nanoparticles (backpacking). Magnetic nanoparticle cell labeling is of particular interest due to opportunities for magnetic separation, in vivo manipulation, drug delivery and magnetic resonance imaging (MRI). While modification of T cells with magnetic nanoparticles (MNPs) was explored before, we questioned whether MNPs are compatible with CAR-T cells when introduced during the manufacturing process. We chose highly aminated 120 nm crosslinked iron oxide nanoworms (CLIO NWs, ~36,000 amines per NW) that could efficiently label different adherent cell lines and we used CD123 CAR-T cells as the labeling model. The CD123 CAR-T cells were produced in the presence of CLIO NWs, CLIO NWs plus protamine sulfate (PS), or PS only. The transduction efficiency of lentiviral CD123 CAR with only NWs was ~23% lower than NW+PS and PS groups (~33% and 35%, respectively). The cell viability from these three transduction conditions was not reduced within CAR-T cell groups, though lower compared to non-transduced T cells (mock T). Use of CLIO NWs instead of, or together with cationic protamine sulfate for enhancement of lentiviral transduction resulted in comparable levels of CAR expression and viability but decreased the proportion of CD8+ cells and increased the proportion of CD4+ cells. CD123 CAR-T transduced in the presence of CLIO NWs, CLIO NWs plus PS, or PS only, showed similar level of cytotoxicity against leukemic cell lines. Furthermore, fluorescence microscopy imaging demonstrated that CD123 CAR-T cells labeled with CLIO NW formed rosettes with CD123+ leukemic cells as the non-labeled CAR-T cells, indicating that the CAR-T targeting to tumor cells has maintained after CLIO NW labeling. The in vivo trafficking of the NW labeled CAR-T cells showed the accumulation of CAR-T labeled with NWs primarily in the bone marrow and spleen. CAR-T cells can be magnetically labeled during their production while maintaining functionality using the positively charged iron oxide NWs, which enable the in vivo biodistribution and tracking of CAR-T cells., Competing Interests: Declaration of Competing Interest The authors declare that they have no known conflict of interest, financial or otherwise.

Umbilical cord blood (UCB) transplantation is a potentially curative treatment for patients with refractory severe aplastic anaemia (SAA), but has historically been associated with delayed engraftment and high graft failure and mortality rates. We conducted a prospective phase 2 trial to assess outcome of an allogeneic transplant regimen that co-infused a single UCB unit with CD34 + -selected cells from a haploidentical relative. Among 29 SAA patients [including 10 evolved to myelodysplastic syndrome (MDS)] who underwent the haplo cord transplantation (median age 20 years), 97% had neutrophil recovery (median 10 days), and 93% had platelet recovery (median 32 days). Early myeloid engraftment was from the haplo donor and was gradually replaced by durable engraftment from UCB in most patients. The cumulative incidences of grade II-IV acute and chronic graft-versus-host disease (GVHD) were 21% and 41%, respectively. With a median follow-up of 7·5 years, overall survival was 83% and GVHD/relapse-free survival was 69%. Patient- and transplant-related factors had no impact on engraftment and survival although transplants with haplo-versus-cord killer-cell immunoglobulin-like receptor (KIR) ligand incompatibility had delayed cord engraftment. Our study shows haplo cord transplantation is associated with excellent engraftment and long-term outcome, providing an alternative option for patients with refractory SAA and hypoplastic MDS who lack human leucocyte antigen (HLA)-matched donors., (© 2021 British Society for Haematology and John Wiley & Sons Ltd. This article has been contributed to by US Government employees and their work is in the public domain in the USA.)

Background: Lisocabtagene maraleucel (liso-cel) is an autologous, CD19-directed, chimeric antigen receptor (CAR) T-cell product. We aimed to assess the activity and safety of liso-cel in patients with relapsed or refractory large B-cell lymphomas., Methods: We did a seamless design study at 14 cancer centres in the USA. We enrolled adult patients (aged ≥18 years) with relapsed or refractory large B-cell lymphomas. Eligible histological subgroups included diffuse large B-cell lymphoma, high-grade B-cell lymphoma with rearrangements of MYC and either BCL2, BCL6, or both (double-hit or triple-hit lymphoma), diffuse large B-cell lymphoma transformed from any indolent lymphoma, primary mediastinal B-cell lymphoma, and follicular lymphoma grade 3B. Patients were assigned to one of three target dose levels of liso-cel as they were sequentially tested in the trial (50 × 10 6 CAR + T cells [one or two doses], 100 × 10 6 CAR + T cells, and 150 × 10 6 CAR + T cells), which were administered as a sequential infusion of two components (CD8 + and CD4 + CAR + T cells) at equal target doses. Primary endpoints were adverse events, dose-limiting toxicities, and the objective response rate (assessed per Lugano criteria); endpoints were assessed by an independent review committee in the efficacy-evaluable set (comprising all patients who had confirmed PET-positive disease and received at least one dose of liso-cel). This trial is registered with ClinicalTrials.gov, NCT02631044., Findings: Between Jan 11, 2016, and July 5, 2019, 344 patients underwent leukapheresis for manufacture of CAR + T cells (liso-cel), of whom 269 patients received at least one dose of liso-cel. Patients had received a median of three (range 1-8) previous lines of systemic treatment, with 260 (97%) patients having had at least two lines. 112 (42%) patients were aged 65 years or older, 181 (67%) had chemotherapy-refractory disease, and seven (3%) had secondary CNS involvement. Median follow-up for overall survival for all 344 patients who had leukapheresis was 18·8 months (95% CI 15·0-19·3). Overall safety and activity of liso-cel did not differ by dose level. The recommended target dose was 100 × 10 6 CAR + T cells (50 × 10 6 CD8 + and 50 × 10 6 CD4 + CAR + T cells). Of 256 patients included in the efficacy-evaluable set, an objective response was achieved by 186 (73%, 95% CI 66·8-78·0) patients and a complete response by 136 (53%, 46·8-59·4). The most common grade 3 or worse adverse events were neutropenia in 161 (60%) patients, anaemia in 101 (37%), and thrombocytopenia in 72 (27%). Cytokine release syndrome and neurological events occurred in 113 (42%) and 80 (30%) patients, respectively; grade 3 or worse cytokine release syndrome and neurological events occurred in six (2%) and 27 (10%) patients, respectively. Nine (6%) patients had a dose-limiting toxicity, including one patient who died from diffuse alveolar damage following a dose of 50 × 10 6 CAR + T cells., Interpretation: Use of liso-cel resulted in a high objective response rate, with a low incidence of grade 3 or worse cytokine release syndrome and neurological events in patients with relapsed or refractory large B-cell lymphomas, including those with diverse histological subtypes and high-risk features. Liso-cel is under further evaluation at first relapse in large B-cell lymphomas and as a treatment for other relapsed or refractory B-cell malignancies., Funding: Juno Therapeutics, a Bristol-Myers Squibb Company., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

We compared outcomes among adult matched related donor (MRD) patients undergoing peripheral blood stem cell transplantation and adult patients undergoing double unit cord blood transplantation (CBT) at our center between 2010 and 2017. A total of 190 CBT patients were compared with 123 MRD patients. Median follow-up was 896 days (range, 169-3350) among surviving CBT patients and 1262 days (range, 249-3327) among surviving MRD patients. Comparing all CBT with all MRD patients, overall survival (OS) was comparable (P = .61) and graft-versus-host disease (GVHD) relapse-free survival (GRFS) was significantly improved among CBT patients (P = .0056), primarily because of decreased moderate to severe chronic GVHD following CBT (P < .0001; hazard ratio [HR], 3.99; 95% confidence interval [CI], 2.26-7.04). Among patients undergoing our most commonly used MRD and umbilical cord blood (CB) myeloablative regimens, OS was comparable (P = .136) and GRFS was significantly improved among CBT patients (P = .006). Cumulative incidence of relapse trended toward decreased in the CBT group (P = .075; HR, 1.85; CI 0.94-3.67), whereas transplant-related mortality (TRM) was comparable (P = .55; HR, 0.75; CI, 0.29-1.95). Among patients undergoing our most commonly used nonmyeloablative regimens, OS and GRFS were comparable (P = .158 and P = .697). Cumulative incidence of both relapse and TRM were comparable (P = .32; HR, 1.35; CI, 0.75-2.5 for relapse and P = .14; HR, 0.482; CI, 0.18-1.23 for TRM). Our outcomes support the efficacy of CBT and suggest that among patients able to tolerate more intensive conditioning regimens at high risk for relapse, CB may be the preferred donor source., (© 2020 by The American Society of Hematology.)

Heavily transfused patients frequently develop human leukocyte antigen (HLA) allo-immunization resulting in platelet transfusion refractoriness and a high risk for life-threatening thrombocytopenia. Data suggest complement activation leading to the destruction of platelets bound by HLA allo-antibodies may play a pathophysiologic role in platelet refractoriness. Here we conducted a pilot trial to investigate the use of eculizumab, a monoclonal antibody that binds and inhibits C5 complement, to treat platelet transfusion refractoriness in allo-immunized patients with severe thrombocytopenia. A single eculizumab infusion was administered to 10 eligible patients, with four (40%) patients overcoming platelet refractories assessed measuring the corrected platelet count increment (CCI) 10-60 min and 18-24 h post transfusion. Responding patients had a reduction in the requirement for subsequent platelet transfusions and had higher post-transfusion platelet increments for 14 days following eculizumab administration. Remarkably, three of the four responders met CCI criteria for response despite receiving HLA-incompatible platelets. Our results suggest that eculizumab has the ability to overcome platelet transfusion refractoriness in patients with broad HLA allo-immunization. This study establishes proof of principle that complement inhibition can treat platelet transfusion refractoriness, laying the foundation for a large multicentre trial to assess the overall efficacy of this approach (ClinicalTrials.gov, identifier: NCT02298933)., (© 2020 British Society for Haematology and John Wiley & Sons Ltd.)

We compared CMV outcomes of three prophylactic approaches used for CBT and haploidentical cord transplants from December 2009 through 2018: letermovir (n = 32) through day 100 post transplant, "valacyclovir day 100" (valacyclovir 2 g orally three times daily through day 100) (n = 60), and "valacyclovir hospital discharge" (valacyclovir 2 g orally three times daily through hospital discharge then acyclovir 800 mg twice daily) (n = 41). Through day 100, none in the letermovir group, six (10%) in the "valacyclovir day 100," and nine (22%) in the "valacyclovir hospital discharge" group required CMV directed treatment (p = 0.005 and 0.06 comparing letermovir to "valacyclovir hospital discharge" and "valacyclovir day 100"). Fewer patients in the letermovir group (n = 7, 22%) had any CMV reactivation versus the "valacyclovir day 100" group (n = 20, 33%) versus the "valacyclovir hospital discharge" group (n = 23, 57%) (p = 0.003 and 0.21 comparing letermovir to "valacyclovir hospital discharge" and "valacyclovir day 100"). Among patients not reactivating CMV before 100 days, reactivation rates between day 100 and 180 were higher in the letermovir and "valacyclovir day 100" groups than the "valacyclovir hospital discharge" group. Letermovir is safe and effective compared with alternative prophylaxis approaches following CBT through day 100. Reactivation and monitoring after day 100 remain potential concerns.

The endophytic fungus Epichloë festucae is known to produce bioactive metabolites, which consequently protect the host plants from biotic and abiotic stresses. We previously found that the overexpression of vibA (a gene for transcription factor) in E. festucae strain E437 resulted in the secretion of an unknown fungicide. In the present study, the active substance was purified and chemically identified as ε-poly-L-lysine (ε-PL), which consisted of 28-34 lysine units. The productivity was 3.7-fold compared with that of the wild type strain E437. The isolated ε-PL showed inhibitory activity against the spore germination of the plant pathogens Drechslera erythrospila , Botrytis cinerea , and Phytophthora infestans at 1-10 μg/mL. We also isolated the fungal gene " epls " encoding ε-PL synthetase Epls. Overexpression of epls in the wild type strain E437 resulted in the enhanced production of ε-PL by 6.7-fold. Interestingly, overexpression of epls in the different strain E. festucae Fl1 resulted in the production of shorter ε-PL with 8-20 lysine, which exhibited a comparable antifungal activity to the longer one. The results demonstrate the first example of ε-PL synthetase gene from the eukaryotic genomes and suggest the potential of enhanced expression of vibA or/and epls genes in the Epichloë endophyte for constructing pest-tolerant plants.

Venetoclax is approved for older untreated acute myeloid leukemia (AML) patients. Venetoclax was available prior to approval off-label. We assessed our single-institution off-label experience with venetoclax/azacitidine, comparing outcomes with a clinical trial cohort that administered this regimen at the same institution. Thirty-three untreated AML patients unfit or unwilling to receive induction chemotherapy and prescribed venetoclax/azacitidine off-trial were retrospectively analyzed and compared with 33 patients who received the same therapy on trial. Outcomes were compared, and comparisons were made to a theoretical scenario in which off-trial patients received induction. Digital droplet polymerase chain reaction evaluated measurable residual disease (MRD). Off-trial venetoclax was attainable in nearly all patients for whom this was desired. The complete remission (CR)/CR with incomplete blood count recovery rate was 63.3% for off-trial patients who received treatment and 84.9% for trial patients (P = .081). The median overall survival for off-trial patients who received treatment was 381 days (95% confidence interval [CI], 174, not reached) vs 880 days (95% CI, 384, not reached) for trial patients (P = .041). Prior exposure to hypomethylating agents was associated with worse outcomes. Response rates with venetoclax/azacitidine were not inferior to a theoretical scenario in which patients received induction, and early death rates were less than expected with induction. MRD negativity was achievable. Newly diagnosed AML patients treated in a "real-world" scenario with off-trial venetoclax/azacitidine had inferior outcomes compared with patients treated in the setting of a clinical trial. Additionally, this therapy may be as effective, and less toxic, when compared with induction chemotherapy., (© 2019 by The American Society of Hematology.)

Cell-based therapeutics are one of the most promising and exciting breakthroughs in modern medicine. Modification of the cell surface with ligands, biologics, drugs, and nanoparticles can further enhance the functionality. Previously, we described the synthesis of a dioctadecyl indocarbocyanine Cy3 analog (aminomethyl-DiI) for efficient and stable modification (painting) of mouse erythrocytes with small molecules, enzymes, and biologics. Here, we synthesized a near-infrared aminomethyl dioctadecyl derivative of Cy7 (aminomethyl-DOCy7) and systematically compared it to aminomethyl-DiI as an anchor for the modification of human erythrocytes, Jurkat cells, and primary T cells with immunoglobulin G. To enable copper-free click chemistry modification of cell membranes, we conjugated a methyltetrazine (MTz) group to the amino-indocyanine lipids via a polyethylene glycol (PEG) linker. DOCy7-PEG3400-MTz showed over 99% modification efficiency of human red blood cells (RBCs) at 25 μM. Reaction of trans -cyclooctene (TCO) modified immunoglobulin G (IgG) with DOCy7-PEG 4 -MTz-modified RBCs (2-step method) resulted in ∼80,000 IgG molecules per erythrocyte, whereas modification with a preconjugated DOCy7-PEG3400-IgG construct (1-step method) resulted in ∼20,000 IgG molecules per erythrocyte as detected by immuno dot-blot. The number of IgG/RBC was controlled by the concentration of IgG. The incubation of RBCs with DiI-PEG3400-MTz resulted in a similar number of IgG/RBC. Modification of the T-lymphocyte cell line Jurkat with IgG resulted in ∼1 × 10 6 IgG/cell with the 1-step and 2-step methods, and the efficiency was similar for DOCy7 and DiI constructs. Finally, we used DOCy7 and DiI constructs to demonstrate efficient modification of primary CD3+T cells from healthy donors. In conclusion, click indocarbocyanine conjugates represent a novel multicolor chemical biology tool kit for efficient surface modification of different cells types and can be used for potential imaging and drug delivery applications involving engineered cells.

Myelodysplastic syndrome (MDS) is a group of heterogeneous disorders caused by ineffective hematopoiesis and characterized by bone marrow dysplasia and cytopenia. Current treatment options for MDS are limited to supportive care, hypomethylating agents, and stem cell transplant. Most patients eventually succumb to the disease or progress to leukemia. Previously, we found that CD123 can be used to delineate MDS stem cells in patients at high risk for MDS and that the CD123-positive population is biologically distinct from normal hematopoietic stem cells. Furthermore, selective targeting of MDS stem cells can dramatically reduce tumor burden in preclinical models. On the basis of these findings, we propose CD123 as a candidate target for chimeric antigen receptor (CAR) T-cell therapy in high-risk MDS patients. To test this concept, we employed a CAR lentiviral vector containing a CD123-specific single-chain variable fragment in combination with the CD28 costimulatory domain, CD3ζ signaling domain, and truncated estimated glomerular filtration rate. Utilizing this system, we illustrate that CD123 CAR can be expressed on both healthy donor and MDS patient-derived T lymphocytes with high efficiency, leading to the successful elimination of MDS stem cells both in vitro and in patient-derived xenografts. These results provide the concept for the use of CD123-targeted CAR T cells as a therapeutic option for patients with MDS., (Copyright © 2019 ISEH -- Society for Hematology and Stem Cells. All rights reserved.)

Acute myeloid leukemia (AML) is the most common acute leukemia in adults. Leukemia stem cells (LSCs) drive the initiation and perpetuation of AML, are quantifiably associated with worse clinical outcomes, and often persist after conventional chemotherapy resulting in relapse 1-5 . In this report, we show that treatment of older patients with AML with the B cell lymphoma 2 (BCL-2) inhibitor venetoclax in combination with azacitidine results in deep and durable remissions and is superior to conventional treatments. We hypothesized that these promising clinical results were due to targeting LSCs. Analysis of LSCs from patients undergoing treatment with venetoclax + azacitidine showed disruption of the tricarboxylic acid (TCA) cycle manifested by decreased α-ketoglutarate and increased succinate levels, suggesting inhibition of electron transport chain complex II. In vitro modeling confirmed inhibition of complex II via reduced glutathionylation of succinate dehydrogenase. These metabolic perturbations suppress oxidative phosphorylation (OXPHOS), which efficiently and selectively targets LSCs. Our findings show for the first time that a therapeutic intervention can eradicate LSCs in patients with AML by disrupting the metabolic machinery driving energy metabolism, resulting in promising clinical activity in a patient population with historically poor outcomes.

From an organismal perspective, cancer cell populations can be considered analogous to parasites that compete with the host for essential systemic resources such as glucose. Here, we employed leukemia models and human leukemia samples to document a form of adaptive homeostasis, where malignant cells alter systemic physiology through impairment of both host insulin sensitivity and insulin secretion to provide tumors with increased glucose. Mechanistically, tumor cells induce high-level production of IGFBP1 from adipose tissue to mediate insulin sensitivity. Further, leukemia-induced gut dysbiosis, serotonin loss, and incretin inactivation combine to suppress insulin secretion. Importantly, attenuated disease progression and prolonged survival are achieved through disruption of the leukemia-induced adaptive homeostasis. Our studies provide a paradigm for systemic management of leukemic disease., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Chronic myeloid leukemia (CML) is defined by the presence of Philadelphia chromosome (Ph), resulting from a reciprocal translocation between chromosomes 9 and 22 [t(9;22] that gives rise to a BCR-ABL1 fusion gene. CML occurs in 3 different phases (chronic, accelerated, and blast phase) and is usually diagnosed in the chronic phase. Tyrosine kinase inhibitor (TKI) therapy is a highly effective first-line treatment option for all patients with newly diagnosed chronic phase CML (CP-CML). The selection TKI therapy should be based on the risk score, toxicity profile of TKI, patient's age, ability to tolerate therapy, and the presence of comorbid conditions. This manuscript discusses the recommendations outlined in the NCCN Guidelines for the diagnosis and management of patients with CP-CML., (Copyright © 2018 by the National Comprehensive Cancer Network.)

The "Minnesota" reduced-intensity conditioning (RIC) cord blood transplantation (CBT) regimen (standard RIC) of fludarabine (Flu) (200 mg/m 2 ), cyclophosphamide (Cy) (50 mg/kg), and 200- or 300-cGy total body irradiation (TBI) is the most published RIC CBT regimen. Though well tolerated, high relapse rates remain a concern with this regimen. Intensification of conditioning may reduce relapse without increasing transplant-related mortality (TRM). We performed a retrospective cohort comparison of outcomes in adult patients who underwent first double-unit CBT with standard RIC as compared with the intensified regimen of Flu 150 mg/m 2 , Cy 50 mg/kg, thiotepa 10 mg/kg, and 400-cGy TBI (intensified RIC). Of the 99 patients studied, 47 received intensified RIC. Acute myelogenous leukemia was the major indication for transplant. The median age at transplant was 67 years (range, 24 to 74 years) and 54 years (range, 25 to 67 years) in standard RIC and intensified RIC, respectively. Median hematopoietic stem cell transplantation comorbidity index was 3 (range, 0 to 5) and 1 (range, 0 to 6) in the standard RIC and intensified RIC groups, respectively. Median follow-up among survivors was 22 months (range, 3.7 to 79 months) following standard RIC and 15 months (range, 2.8 to 36 months) following intensified RIC. The cumulative incidence (CI) of relapse was significantly lower following intensified RIC compared with standard RIC (P = .0013); this finding maintained significance in multivariate analysis (P = .045). TRM was comparable between the 2 groups (P = .99). Overall survival (OS) was significantly improved following intensified RIC as compared with standard RIC (P = .03). Median OS was 17 months following standard RIC versus not reached followed intensified RIC. The CI of grade II to IV acute graft-versus-host disease (GVHD) was significantly higher in the intensified RIC cohort than the standard RIC-cohort (P = .007), while CI of grade III to IV acute GVHD, any chronic GVHD, and moderate-to-severe chronic GVHD was comparable in each cohort (P = .20, P = .21, and P = .61, respectively). This retrospective analysis shows an improvement in OS and decreased relapse without increase in TRM in patients receiving intensified RIC as compared with standard RIC. Our data suggest that consideration of thiotepa-based intensified RIC may improve outcomes in fit, older patients undergoing double-unit CBT., (Copyright © 2018 The American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

Hematopoietic stem cells can be mobilized from healthy donors using single-agent plerixafor without granulocyte colony-stimulating factor and, following allogeneic transplantation, can result in sustained donor-derived hematopoiesis. However, when a single dose of plerixafor is administered at a conventional 240 μg/kg dose, approximately one-third of donors will fail to mobilize the minimally acceptable dose of CD34 + cells needed for allogeneic transplantation. We conducted an open-label, randomized trial to assess the safety and activity of high-dose (480 μg/kg) plerixafor in CD34 + cell mobilization in healthy donors. Subjects were randomly assigned to receive either a high dose or a conventional dose (240 μg/kg) of plerixafor, given as a single subcutaneous injection, in a two-sequence, two-period, crossover design. Each treatment period was separated by a 2-week minimum washout period. The primary endpoint was the peak CD34 + count in the blood, with secondary endpoints of CD34 + cell area under the curve (AUC), CD34 + count at 24 hours, and time to peak CD34 + following the administration of plerixafor. We randomized 23 subjects to the two treatment sequences and 20 subjects received both doses of plerixafor. Peak CD34 + count in the blood was significantly increased (mean 32.2 versus 27.8 cells/μL, P =0.0009) and CD34 + cell AUC over 24 hours was significantly increased (mean 553 versus 446 h cells/μL, P <0.0001) following the administration of the 480 μg/kg dose of plerixafor compared with the 240 μg/kg dose. Remarkably, of seven subjects who mobilized poorly (peak CD34 + ≤20 cells/μL) after the 240 μg/kg dose of plerixafor, six achieved higher peak CD34 + cell numbers and all achieved higher CD34 + AUC over 24 hours after the 480 μg/kg dose. No grade 3 or worse drug-related adverse events were observed. This study establishes that high-dose plerixafor can be safely administered in healthy donors and mobilizes greater numbers of CD34 + cells than conventional-dose plerixafor, which may improve CD34 + graft yields and reduce the number of apheresis procedures needed to collect sufficient stem cells for allogeneic transplantation. ( ClinicalTrials.gov, identifier: NCT00322127 )., (Copyright© Ferrata Storti Foundation.)

Allogeneic haematopoietic stem cell transplantation is curative for severe aplastic anaemia (SAA) unresponsive to immunosuppressive therapy. To reduce chronic graft-versus-host disease (GVHD), which occurs more frequently after peripheral blood stem cell (PBSC) transplantation compared to bone-marrow transplantation (BMT), and to prevent graft rejection, we developed a novel partial T-cell depleted transplant that infuses high numbers of granulocyte colony-stimulating factor-mobilized CD34 + selected PBSCs combined with a BMT-equivalent dose of non-mobilized donor T-cells. Fifteen patients with refractory SAA received cyclophosphamide, anti-thymocyte globulin and fludarabine conditioning, and were transplanted with a median 8 × 10 6 CD34 +  cells/kg and 2 × 10 7 non-mobilized CD3 + T-cells/kg from human leucocyte antigen-matched sibling donors. All achieved sustained engraftment with only two developing acute and two developing chronic GVHD. With a 3·5-year median follow-up, 86% of patients survived and were transfusion-independent. When compared to a retrospective cohort of 56 bone-marrow failure patients that received the identical transplant preparative regimen and GVHD prophylaxis with the exception that the allograft contained unmanipulated PBSCs, partial T-cell depleted transplant recipients had delayed donor T-cell chimerism and relative reduction of 75% in the incidence of acute grade II-IV GVHD (13% vs. 52%; P = 0·010) and of 82% in chronic GVHD (13% vs. 72%; P = 0·0004). In multivariate analysis, partial T-cell depleted transplants remained significantly associated with a reduced risk of GVHD. In conclusion, for patients with refractory SAA, this novel transplant strategy achieves excellent engraftment and survival when compared to unmanipulated PBSC transplants and dramatically reduces the incidence of both acute and chronic GVHD., (© 2017 John Wiley & Sons Ltd.)

The NCCN Guidelines for Chronic Myeloid Leukemia (CML) provide recommendations for the management of chronic-phase and advanced-phase CML in adult patients. The median age of disease onset is 67 years. However, because CML occurs in all age groups, clinical care teams should be prepared to address issues relating to fertility and pregnancy with patients who are of reproductive age at the time of diagnosis. CML is relatively rare in children and there are no evidence-based recommendations for the management of CML in pediatric population. These NCCN Guidelines Insights discuss special considerations for the management of CML during pregnancy and for the management of CML in the pediatric population., (Copyright © 2016 by the National Comprehensive Cancer Network.)

We report a patient with paramyotonia congenita/hyperkalemic periodic paralysis due to Nav1.4 I693T mutation who had worsening of myotonia and muscle weakness in the setting of hypomagnesemia and hypocalcemia with marked recovery after magnesium administration. Computer simulations of the effects of the I693T mutation were introduced in the muscle fiber model by both hyperpolarizing shifts in the Nav1.4 channel activation and a faster recovery from slow channel inactivation. A further shift in the Nav1.4 channel activation in the hyperpolarizing direction as expected with low divalent cations resulted in myotonia that progressed to membrane inexcitability. Shifting the channel activation in the depolarizing direction as would be anticipated from magnesium supplementation abolished the myotonia. These observations provide clinical and biophysical evidence that the muscle symptoms in sodium channelopathy are sensitive to divalent cations. Exploration of the role of magnesium administration in therapy or prophylaxis is warranted with a randomized clinical trial., (Published by Elsevier B.V.)

The anti-invasive and anti-proliferative effects of betulins and abietane derivatives was systematically tested using an organotypic model system of advanced, castration-resistant prostate cancers. A preliminary screen of the initial set of 93 compounds was performed in two-dimensional (2D) growth conditions using non-transformed prostate epithelial cells (EP156T), an androgen-sensitive prostate cancer cell line (LNCaP), and the castration-resistant, highly invasive cell line PC-3. The 25 most promising compounds were all betulin derivatives. These were selected for a focused secondary screen in three-dimensional (3D) growth conditions, with the goal to identify the most effective and specific anti-invasive compounds. Additional sensitivity and cytotoxicity tests were then performed using an extended cell line panel. The effects of these compounds on cell cycle progression, mitosis, proliferation and unspecific cytotoxicity, versus their ability to specifically interfere with cell motility and tumor cell invasion was addressed. To identify potential mechanisms of action and likely compound targets, multiplex profiling of compound effects on a panel of 43 human protein kinases was performed. These target de-convolution studies, combined with the phenotypic analyses of multicellular organoids in 3D models, revealed specific inhibition of AKT signaling linked to effects on the organization of the actin cytoskeleton as the most likely driver of altered cell morphology and motility.

We report a case of t(8;21) acute myeloid leukemia presenting as severe aplastic anemia. While initial bone marrow biopsy lacked any cytogenetic abnormalities in 20 analyzed metaphases, repeat bone marrow biopsy eight days later demonstrated this translocation. Initial cytogenetic analysis of 20 metaphases was therefore insufficient to make the diagnosis of hypocellular acute myeloid leukemia. We discuss that further complementary molecular tests, such as CGH, would likely provide a more robust diagnosis of hematopoietic diseases.

One of the mechanisms by which all-trans retinoic acid (ATRA) has been shown to suppress the growth of CAOV3 ovarian carcinoma cells involves an increase in the accumulation of Rb2/p130 protein, a member of the retinoblastoma family of tumor suppressors. This increase in accumulation of RB2/p130 by ATRA results from increased stability of Rb2/p130 protein as a result of an increase in dephosphorylation of the protein by the serine/threonine phosphatase PP2A. We show that upon ATRA treatment, PP2A interacts with the Rb2/p130 C-terminus and specifically dephosphorylates two residues (S1080 and T1097) adjacent to NLS1 and NLS2 of Rb2/p130. Moreover, co-immunoprecipitation studies reveal that Rb2/p130 can form a complex with the nuclear transport proteins, importin α and importin β, binding to the same dephosphorylated NLS1 and NLS2 sites. Finally, mutation of S1080 and T1097 results in retension of Rb2/p130 in the cytoplasm. Our studies suggest that one mechanism by which ATRA treatment of CAOV3 cells induces G0/G1 arrest involves the recruitment of PP2A to the C-terminus of Rb2/p130, resulting in the dephosphorylation of the S1080 and T1097 adjacent to the NLS and the subsequent interaction of Rb2/p130 with importins leading to transport of the Rb2/p130 to the nucleus where it inhibits cell-cycle progression., (Copyright © 2010 Wiley-Liss, Inc.)

c-Cbl and Cbl-b are highly conserved adaptor proteins that participate in integrin signaling, regulating cytoskeletal organization, motility, and bone resorption. Deletion of both c-Cbl and Cbl-b in mice leads to embryonic lethality, indicating that the two proteins perform essential redundant functions. To examine the redundant actions of c-Cbl and Cbl-b in osteoclasts, we depleted c-Cbl in Cbl-b(-/-) osteoclasts by using a short hairpin RNA. Depleting both Cbl proteins disrupted both the podosome belt and the microtubule network and decreased bone-resorbing activity. Stabilizing the microtubules with paclitaxel or inhibiting histone deacetylase 6 (HDAC6), which destabilizes microtubules by deacetylating beta-tubulin, protected both the microtubule network and the podosome belt. Examination of the mechanism involved demonstrated that the conserved four-helix bundle of c-Cbl's tyrosine kinase binding domain bound to beta-tubulin, and both c-Cbl and Cbl-b displaced HDAC6. In addition to the effects on microtubules and the podosome belt, depleting both Cbls significantly increased the levels of the proapoptotic protein Bim and apoptosis relative to the levels induced by eliminating either protein alone. Thus, both c-Cbl and Cbl-b promote bone resorption via the stabilization of microtubules, allowing the formation of the podosome belt in osteoclasts, and by promoting osteoclast survival.

Cbl is an adaptor protein and ubiquitin ligase that binds and is phosphorylated by the nonreceptor tyrosine kinase Src. We previously showed that the primary interaction between Src and Cbl is mediated by the Src homology domain 3 (SH3) of Src binding to proline-rich sequences of Cbl. The peptide Cbl RDLPPPPPPDRP(540-551), which corresponds to residues 540-551 of Cbl, inhibited the binding of a GST-Src SH3 fusion protein to Cbl, whereas RDLAPPAPPPDR(540-551) did not, suggesting that Src binds to this site on Cbl in a class I orientation. Mutating prolines 543-548 reduced Src binding to the Cbl 479-636 fragment significantly more than mutating the prolines in the PPVPPR(494-499) motif, which was previously reported to bind Src SH3. Mutating Cbl prolines 543-548 to alanines substantially reduced Src binding to Cbl, Src-induced phosphorylation of Cbl, and the inhibition of Src kinase activity by Cbl. Expressing the mutated Cbl in osteoclasts induced a moderate reduction in bone-resorbing activity and increased amounts of Src protein. In contrast, disabling the tyrosine kinase-binding domain of full-length Cbl by mutating glycine 306 to glutamic acid, and thereby preventing the previously described binding of the tyrosine kinase-binding domain to the Src phosphotyrosine 416, had no effect on Cbl phosphorylation, the inhibition of Src activity by full-length Cbl, or bone resorption. These data indicate that the Cbl RDLPPPP(540-546) sequence is a functionally important binding site for Src.

High-grade gliomas are devastating brain tumors associated with a mean survival of <50 weeks. Two of the most common genetic changes observed in these tumors are overexpression/mutation of the epidermal growth factor receptor (EGFR) vIII and loss of PTEN/MMAC1 expression. To determine whether somatically acquired EGFRvIII expression or Pten loss accelerates high-grade glioma development, we used a previously characterized RasB8 glioma-prone mouse strain, in which these specific genetic changes were focally introduced at 4 weeks of age. We show that both postnatal EGFRvIII expression and Pten inactivation in RasB8 mice potentiate high-grade glioma development. Moreover, we observe a concordant loss of Pten and EGFR overexpression in nearly all high-grade gliomas induced by either EGFRvIII introduction or Pten inactivation. This novel preclinical model of high-grade glioma will be useful in evaluating brain tumor therapies targeted to the pathways specifically dysregulated by EGFR expression or Pten loss.

Purpose: The gastrointestinal carcinoma-associated antigen epithelial cell adhesion molecule (EpCAM) has been a target for passive and active immunotherapy of gastrointestinal carcinoma patients. The antigen is expressed by both tumor and normal tissues. The immunogenicity of EpCAM in colorectal cancer patients has been described previously. The purpose of this study was to evaluate humoral and cellular immune responses of healthy individuals and ulcerative colitis patients to EpCAM and to relate immune responses to colonic tissue expression of EpCAM., Methods: An inhibition radioimmunoassay was used to detect anti-EpCAM serum antibodies. Anti-EpCAM antibodies of a healthy donor were expressed by phages and sequenced. (3)H-thymidine incorporation assay was used for detection of lymphoproliferative responses to stimulation with EpCAM. EpCAM tissue expression was determined by immunohistochemistry., Results: We detected anti-EpCAM serum antibodies in 4 of 10, and EpCAM-specific lymphoproliferation responses in 1 of 10 healthy volunteers. The majority of anti-EpCAM antibodies derived from a healthy donor were germline-encoded. In contrast, none of the 23 patients with ulcerative colitis showed serum antibodies to EpCAM (P=0.005). Antigen expression was greatly reduced and altered in ulcerative colitis patients, whereas colon from healthy individuals and uninvolved colon of colorectal cancer patients expressed high levels of EpCAM., Conclusion: The results of these studies suggest an association between EpCAM antibody production and colonic EpCAM expression in healthy individuals and patients with ulcerative colitis. Decreased and altered colonic EpCAM expression in ulcerative colitis patients may be related to the disease induction, based on the previously demonstrated adhesion function of this molecule. Healthy individuals with anti-EpCAM immune responses and high risk for developing colorectal carcinoma are prime candidates for prophylactic immunization against EpCAM.

All-trans retinoic acid (ATRA) treatment causes CAOV3 ovarian carcinoma cells to growth arrest in the G0/G1 phase and to elevate the level of Rb2/p130 protein. PP2A, a serine/threonine phosphatase, binds and dephosphorylates Rb2/p130, thereby increasing the half-life of Rb2/p130 in the cell. In order to further characterize the interaction between Rb2/p130 and PP2A upon ATRA treatment, we examined the posttranslational modification of PP2A. ATRA treatment leads to hypophosphorylation of PP2A catalytic subunit (PP2Ac) that correlates with increased PP2A activity. In addition, the N-terminus of PP2Ac binds directly to NLS sequences located in the C-terminus of Rb2/p130. Furthermore, CAOV3 cells transfected with a truncated Rb2/p130 construct consisting of only the wt C-terminus grew more aggressively and were less sensitive to ATRA treatment when compared to parental CAOV3 cells. In contrast, CAOV3 cells transfected with a truncated Rb2/p130 construct consisting of only the C-terminus in which the NLS sites were mutated and which could not interact with PP2A, were as sensitive to ATRA treatment as parental CAOV3 cells. These studies suggest that ATRA treatment suppresses the growth of CAOV3 cells via a novel posttranscriptional mechanism involving PP2A., (Copyright (c) 2005 Wiley-Liss, Inc.)

To determine whether clonally expanded T cells are present in tumor specimens from patients with epithelial ovarian carcinoma (EOC) we amplified by the non-palindromic adaptor PCR (NPA-PCR) or by Vbeta-specific PCR beta-chain T-cell receptor (TCR) transcripts from these tumor specimens. The amplified transcripts were cloned and sequenced. Sequence analysis revealed the presence of substantial proportions of multiple identical copies of beta-chain TCR transcripts, suggesting the presence of clonal expansions of T cells in these patients, which were statistically significant by the binomial distribution in seven of nine patients. Independent amplification in separate experiments of beta-chain TCR transcripts from one patient by either NPA-PCR or by Vbeta-specific PCR, followed by cloning and sequencing, revealed identical clonal expansions irrespectively of the amplification method used. Multiple identical copies of beta-chain TCR transcripts can be derived only by specific antigen-driven proliferation and clonal expansion of the T-cell clones which recognize these antigens. Because of the very large size of the TCR repertoire, the probability of finding by chance multiple identical copies of these transcripts within an independent sample of T cells is negligible. These results demonstrate that T cells infiltrating solid tumor specimens or malignant ascites of patients with EOC contain monoclonal/oligoclonal populations of T cells.