STAT3 Role in T-Cell Memory Formation.

Along with the clinical success of immuno-oncology drugs and cellular therapies, T-cell biology has attracted considerable attention in the immunology community. Long-term immunity, traditionally analyzed in the context of infection, is increasingly studied in cancer. Many signaling pathways, transcription factors, and metabolic regulators have been shown to participate in the formation of memory T cells. There is increasing evidence that the signal transducer and activator of transcription-3 (STAT3) signaling pathway is crucial for the formation of long-term T-cell immunity capable of efficient recall responses. In this review, we summarize what is currently known about STAT3 role in the context of memory T-cell formation and antitumor immunity.

Systemic Endothelial Activation Is Associated With Early Acute Respiratory Distress Syndrome in Children With Extrapulmonary Sepsis.

OBJECTIVES: Systemic endothelial activation may contribute to sepsis-associated organ injury, including acute respiratory distress syndrome. We hypothesized that children with extrapulmonary sepsis with versus without acute respiratory distress syndrome would have plasma biomarkers indicative of increased endothelial activation and that persistent biomarker changes would be associated with poor outcome. DESIGN: Observational cohort. SETTING: Academic PICU. PATIENTS: Patients less than 18 years old with sepsis from extrapulmonary infection with (n = 46) or without (n = 54) acute respiratory distress syndrome and noninfected controls (n = 19). INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Endothelial (angiopoietin-1, angiopoietin-2, tyrosine kinase with immunoglobulin-like loop epidermal growth factor homology domain 2, vascular endothelial growth factor, soluble fms-like tyrosine kinase, von Willebrand factor, E-selectin, intercellular adhesion molecule, vascular cell adhesion molecule, thrombomodulin) and inflammatory biomarkers (C-reactive protein, interleukin-6, and interleukin-8) were measured from peripheral plasma collected within 3 days (time 1) of sepsis recognition and at 3-6 days (time 2) and 7-14 days (time 3). Time 1 biomarkers and longitudinal measurements were compared for sepsis patients with versus without acute respiratory distress syndrome and in relation to complicated course, defined as greater than or equal to two organ dysfunctions at day 7 or death by day 28. Angiopoietin-2, angiopoietin-2/angiopoietin-1 ratio, tyrosine kinase with immunoglobulin-like loop epidermal growth factor homology domain 2, vascular endothelial growth factor, von Willebrand factor, E-selectin, intercellular adhesion molecule, vascular cell adhesion molecule, thrombomodulin, endocan, C-reactive protein, interleukin-6, and interleukin-8 were different between sepsis and noninfected control patients at time 1. Among patients with sepsis, those with acute respiratory distress syndrome had higher angiopoietin-2/angiopoietin-1 ratio, vascular endothelial growth factor, vascular cell adhesion molecule, thrombomodulin, endocan, interleukin-6, and interleukin-8 than those without acute respiratory distress syndrome (all p < 0.003). Angiopoietin-2 and angiopoietin-2/angiopoietin-1 ratio remained higher in sepsis with versus without acute respiratory distress syndrome after multivariable analyses. Time 1 measures of angiopoietin-2, angiopoietin-2/-1 ratio, von Willebrand factor, and endocan were indicative of complicated course in all sepsis patients (all area under the receiver operating curve ≥ 0.80). In sepsis without acute respiratory distress syndrome, soluble fms-like tyrosine kinase decreased more quickly and von Willebrand factor and thrombomodulin decreased more slowly in those with complicated course. CONCLUSIONS: Children with extrapulmonary sepsis with acute respiratory distress syndrome had plasma biomarkers indicative of greater systemic endothelial activation than those without acute respiratory distress syndrome. Several endothelial biomarkers measured near sepsis recognition were associated with complicated course, whereas longitudinal biomarker changes yielded prognostic information only in those without sepsis-associated acute respiratory distress syndrome.

Dominant-Negative TGF-ß Receptor Enhances PSMA-Targeted Human CAR T Cell Proliferation And Augments Prostate Cancer Eradication.

Cancer has an impressive ability to evolve multiple processes to evade therapies. While immunotherapies and vaccines have shown great promise, particularly in certain solid tumors such as prostate cancer, they have been met with resistance from tumors that use a multitude of mechanisms of immunosuppression to limit effectiveness. Prostate cancer, in particular, secretes transforming growth factor ß (TGF-ß) as a means to inhibit immunity while allowing for cancer progression. Blocking TGF-ß signaling in T cells increases their ability to infiltrate, proliferate, and mediate antitumor responses in prostate cancer models. We tested whether the potency of chimeric antigen receptor (CAR) T cells directed to prostate-specific membrane antigen (PSMA) could be enhanced by the co-expression of a dominant-negative TGF-ßRII (dnTGF-ßRII). Upon expression of the dominant-negative TGF-ßRII in CAR T cells, we observed increased proliferation of these lymphocytes, enhanced cytokine secretion, resistance to exhaustion, long-term in vivo persistence, and the induction of tumor eradication in aggressive human prostate cancer mouse models. Based on our observations, we initiated a phase I clinical trial to assess these CAR T cells as a novel approach for patients with relapsed and refractory metastatic prostate cancer (ClinicalTrials.gov: NCT03089203).

Ibrutinib enhances chimeric antigen receptor T-cell engraftment and efficacy in leukemia.

Anti-CD19 chimeric antigen receptor (CAR) T-cell therapy is highly promising but requires robust T-cell expansion and engraftment. A T-cell defect in chronic lymphocytic leukemia (CLL) due to disease and/or therapy impairs ex vivo expansion and response to CAR T cells. To evaluate the effect of ibrutinib treatment on the T-cell compartment in CLL as it relates to CAR T-cell generation, we examined the phenotype and function of T cells in a cohort of CLL patients during their course of treatment with ibrutinib. We found that ≥5 cycles of ibrutinib therapy improved the expansion of CD19-directed CAR T cells (CTL019), in association with decreased expression of the immunosuppressive molecule programmed cell death 1 on T cells and of CD200 on B-CLL cells. In support of these findings, we observed that 3 CLL patients who had been treated with ibrutinib for ≥1 year at the time of T-cell collection had improved ex vivo and in vivo CTL019 expansion, which correlated positively together and with clinical response. Lastly, we show that ibrutinib exposure does not impair CAR T-cell function in vitro but does improve CAR T-cell engraftment, tumor clearance, and survival in human xenograft models of resistant acute lymphocytic leukemia and CLL when administered concurrently. Our collective findings indicate that ibrutinib enhances CAR T-cell function and suggest that clinical trials with combination therapy are warranted. Our studies demonstrate that improved T-cell function may also contribute to the efficacy of ibrutinib in CLL. These trials were registered at www.clinicaltrials.gov as #NCT01747486, #NCT01105247, and #NCT01217749.

Bone marrow mesenchymal stromal cells to treat tissue damage in allogeneic stem cell transplant recipients: correlation of biological markers with clinical responses.

Bone marrow mesenchymal stromal cells (BMSCs) have been used to treat acute graft-versus-host disease (GVHD) and other complications following allogeneic hematopoietic stem cell transplantation (SCT). We conducted a phase I trial using third party, early passage BMSCs for patients with steroid-refractory GVHD, tissue injury, or marrow failure following SCT to investigate safety and efficacy. To identify mechanisms of BMSC immunomodulation and tissue repair, patients were serially monitored for plasma GVHD biomarkers, cytokines, and lymphocyte phenotype. Ten subjects were infused a fixed dose of 2 × 10(6) BMSCs/kg intravenously weekly for three doses. There was no treatment-related toxicity (primary endpoint). Eight subjects were evaluable for response at 4 weeks after the last infusion. Five of the seven patients with steroid-refractory acute GVHD achieved a complete response, two of two patients with tissue injury (pneumomediastinum/pneumothorax) achieved resolution but there was no response in two subjects with delayed marrow failure. Rapid reductions in inflammatory cytokines were observed. Clinical responses correlated with a fall in biomarkers (Reg 3α, CK18, and Elafin) relevant for the site of GVHD or tissue injury. The GVHD complete responders survived significantly longer and had higher baseline absolute lymphocyte and central memory CD4 and CD8 counts. Cytokine changes also segregated with survival. These results confirm that BMSCs are associated with rapid clinical and biomarker responses in GVHD and tissue injury. However, BMSCs were ineffective in patients with prolonged GVHD with lower lymphocyte counts, which suggest that effective GVHD control by BMSCs requires a relatively intact immune system.

Ultra-low dose interleukin-2 promotes immune-modulating function of regulatory T cells and natural killer cells in healthy volunteers.

Low-dose interleukin-2 (IL-2) expands regulatory T cells (Tregs) and natural killer (NK) cells after stem cell transplantation (SCT) and may reduce graft-versus-host disease (GVHD). We hypothesized that ultra-low dose (ULD) IL-2 could serve as an immune-modulating agent for stem cell donors to prevent GVHD following SCT. However, the safety, dose level, and immune signatures of ULD IL-2 in immune-competent healthy subjects remain unknown. Here, we have characterized the phenotype and function of Tregs and NK cells as well as the gene expression and cytokine profiles of 21 healthy volunteers receiving 50,000 to 200,000 units/m(2)/day IL-2 for 5 days. ULD IL-2 was well tolerated and induced a significant increase in the frequency of Tregs with increased suppressive function. There was a marked expansion of CD56(bright) NK cells with enhanced interferon-γ (IFN-γ) production. Serum cytokine profiling demonstrated increase of IFN-γ induced protein 10 (IP-10). Gene expression analysis revealed significant changes in a highly restricted set of genes, including FOXP3, IL-2RA, and CISH. This is the first study to evaluate global immune-modulating function of ULD IL-2 in healthy subjects and to support the future studies administrating ULD IL-2 to stem cell donors.

Allogeneic CAR T Cells: Complex Cellular Therapy Designs Test the Limits of Our Preclinical Models.

All chimeric antigen receptor (CAR) T-cell products currently approved by the FDA are autologous, which poses several challenges for widespread use. In this issue, Degagné and colleagues present their preclinical research on creating off-the-shelf CAR T cells for multiple myeloma. They utilized the CRISPR/Cas12a genome editing platform and gene knock-in techniques to eliminate alloreactivity and decrease susceptibility to natural killer (NK)-cell elimination. This work has led to an ongoing phase I trial of off-the-shelf CAR T cells for multiple myeloma treatment. See related article by Degagné et al., p. 462 (2).

Autologous human preclinical modeling of melanoma interpatient clinical responses to immunotherapeutics.

BACKGROUND: Despite recent advances in immunotherapy, a substantial population of late-stage melanoma patients still fail to achieve sustained clinical benefit. Lack of translational preclinical models continues to be a major challenge in the field of immunotherapy; thus, more optimized translational models could strongly influence clinical trial development. To address this unmet need, we designed a preclinical model reflecting the heterogeneity in melanoma patients' clinical responses that can be used to evaluate novel immunotherapies and synergistic combinatorial treatment strategies. Using our all-autologous humanized melanoma mouse model, we examined the efficacy of a novel engineered interleukin 2 (IL-2)-based cytokine variant immunotherapy. METHODS: To study immune responses and antitumor efficacy for human melanoma tumors, we developed an all-autologous humanized melanoma mouse model using clinically annotated, matched patient tumor cells and peripheral blood mononuclear cells (PBMCs). After inoculating immunodeficient NSG mice with patient tumors and an adoptive cell transfer of autologous PBMCs, mice were treated with anti-PD-1, a novel investigational engineered IL-2-based cytokine (nemvaleukin), or recombinant human IL-2 (rhIL-2). The pharmacodynamic effects and antitumor efficacy of these treatments were then evaluated. We used tumor cells and autologous PBMCs from patients with varying immunotherapy responses to both model the diversity of immunotherapy efficacy observed in the clinical setting and to recapitulate the heterogeneous nature of melanoma. RESULTS: Our model exhibited long-term survival of engrafted human PBMCs without developing graft-versus-host disease. Administration of an anti-PD-1 or nemvaleukin elicited antitumor responses in our model that were patient-specific and were found to parallel clinical responsiveness to checkpoint inhibitors. An evaluation of nemvaleukin-treated mice demonstrated increased tumor-infiltrating CD4+ and CD8+ T cells, preferential expansion of non-regulatory T cell subsets in the spleen, and significant delays in tumor growth compared with vehicle-treated controls or mice treated with rhIL-2. CONCLUSIONS: Our model reproduces differential effects of immunotherapy in melanoma patients, capturing the inherent heterogeneity in clinical responses. Taken together, these data demonstrate our model's translatability for novel immunotherapies in melanoma patients. The data are also supportive for the continued clinical investigation of nemvaleukin as a novel immunotherapeutic for the treatment of melanoma.

Donor lymphocyte count and thymic activity predict lymphocyte recovery and outcomes after matched-sibling hematopoietic stem cell transplant.

Delayed immune recovery is a characteristic feature of allogeneic hematopoietic stem cell transplantation in adult recipients. Although recipient thymic T-cell neogenesis contributes to T-cell regeneration after transplantation, thymic recovery in the transplant recipient decreases with increasing age, and is diminished by intensive preconditioning regimens and graft-versus-host disease. In adult recipients, most events that determine transplant success or failure occur during the period when the majority of circulating T cells is derived from the donor's post thymic T-cell repertoire. As a result, the make-up of the donor lymphocyte compartment may strongly influence immune recovery and transplant outcomes. The aim of this study was to examine donor lymphocyte counts in a series of patients undergoing an allogeneic hematopoietic stem cell transplant to identify the potential contribution of donor regulatory and conventional T lymphocyte populations to immune recovery and transplant outcomes. We examined donor lymphocyte subset counts in relation to post-transplant lymphocyte recovery and transplant events in 220 consecutive myeloablative, T-cell-depleted, HLA-identical sibling hematopoietic stem cell transplant recipients with hematologic malignancies. In a multivariate analysis, absolute numbers of donor CD4(+) recent thymic emigrants were associated with overall survival (P=0.032). The donors' absolute lymphocyte count and thymic production of regulatory T cells were both associated with extensive chronic graft-versus-host disease (P=0.002 and P=0.022, respectively). In conclusion, these results identify donor immune characteristics that are associated with lymphocyte recovery, extensive chronic graft-versus-host disease, and survival in the recipient following allogeneic hematopoietic stem cell transplantation. The study reported here was performed using peripheral blood samples drawn from donors and patients enrolled in the ClinicalTrials.gov-registered trials NCT00001623, NCT00001873, NCT00353860, NCT00066300, NCT00079391, and NCT00398346.

Challenges and opportunities of CAR T-cell therapies for CLL.

Chimeric antigen receptor (CAR) T-cell therapies have transformed the treatment landscape of blood cancers. These engineered receptors which endow T cells with antibody-like target cell recognition combined with the typical T cell target cell lysis abilities. Introduced into the clinic in the 2010s, CAR T-cells have shown efficacy in chronic B lymphocytic leukemia (CLL), but a majority of patients do not achieve sustained remission. Here we discuss the current treatment landscape in CLL using small molecules and allogeneic stem cell transplantation, the niche CAR T-cells filled in this context, and what we have learned from biomarker and mechanistic studies. Several product parameters and improvements are introduced as examples of how the bedside-to-bench is translated into improved CAR T-cells for CLL. We hope to convey to our readers the crucial role translational medicine plays in transforming the treatment outcomes for patients with CLL and how this line of research is an essential component of modern medicine.

Spatial immunosampling of MRI-defined glioblastoma regions reveals immunologic fingerprint of non-contrast enhancing, infiltrative tumor margins.

Glioblastoma (GBM) treatment includes maximal safe resection of the core and MRI contrast-enhancing (CE) tumor. Complete resection of the infiltrative non-contrast-enhancing (NCE) tumor rim is rarely achieved. We established a safe, semi-automated workflow for spatially-registered sampling of MRI-defined GBM regions in 19 patients with downstream analysis and biobanking, enabling studies of NCE, wherefrom recurrence/progression typically occurs. Immunophenotyping revealed underrepresentation of myeloid cell subsets and CD8+ T cells in the NCE. While NCE T cells phenotypically and functionally resembled those in matching CE tumor, subsets of activated (CD69hi) effector memory CD8+ T cells were overrepresented. Contrarily, CD25hi Tregs and other subsets were underrepresented. Overall, our study demonstrated that MRI-guided, spatially-registered, intraoperative immunosampling is feasible as part of routine GBM surgery. Further elucidation of the shared and spatially distinct microenvironmental biology of GBM will enable development of therapeutic approaches targeting the NCE infiltrative tumor to decrease GBM recurrence.

Cytopenia and leukocyte recovery shape cytokine fluctuations after myeloablative allogeneic hematopoietic stem cell transplantation.

BACKGROUND: Allogeneic hematopoietic stem cell transplantation is associated with profound changes in levels of various cytokines. Emphasis has been placed on conditioning-associated mucosal damage and neutropenia and associated bacterial translocation as the initiating conditions predisposing to acute graft-versus-host disease. The post-transplant period is, however, also associated with increases in certain homeostatic cytokines. It is unclear how much the homeostatic drive to lymphocyte recovery and the production of cytokines from the engrafting donor immune system determine cytokine fluctuations in the peri- and immediate post-transplant period. The aim of this study was to examine the contributions of the conditioning regimen, donor engraftment, infections, and graft-versus-host disease to fluctuations in cytokines involved in homeostasis and inflammation. DESIGN AND METHODS: We examined the levels of 33 cytokines in relation to peri- and post-transplant events such as conditioning regimen, chimerism, and acute graft-versus-host disease in myeloablative, non-T cell-replete HLA-identical sibling donor stem cell transplantation for hematologic malignancies. RESULTS: We identified two cytokine storms. The first occurred following conditioning and reached peak levels when all the leukocytes were at their lowest concentrations. The second cytokine storm occurred concurrently with hematopoietic reconstitution and subsided with the achievement of full donor lymphocyte chimerism. CONCLUSIONS: Our results indicate that both recipient-related and donor-related factors contribute to the changes in cytokine levels in the recipient following allogeneic hematopoietic stem cell transplantation. The study reported here was performed using plasma samples drawn from patients enrolled in the ClinicalTrials.gov-registered trials NCT00467961 and NCT00378534.

Adaptable Leukemia Cells Resisting CAR T-cell Attack via B-cell Activation.

Chimeric antigen receptor (CAR) T-cell therapy has achieved remarkable milestones in the treatment of B-cell malignancies. However, cancer cells frequently survive CAR T-cell killing in a large cohort of patients. Relapse oftentimes is associated with antigen loss. In this issue, Im and colleagues report a new mechanism of leukemic-cell resistance to anti-CD19 CAR T cells: Leukemic cells can enable a B-cell activation and germinal center reaction signature, which causes CD19 transcriptional downregulation and survival from CAR exposure. See related article by Im et al., p. 1055 (5).

B cell targeting in CAR T cell therapy: Side effect or driver of CAR T cell function?

Chimeric antigen receptor (CAR) T cell therapies targeting CD19 and CD22 have been successful for treating B cell cancers, but CAR T cells targeting non-B cell cancers remain unsuccessful. We propose that rather than being strictly a side effect of therapy, the large number of CAR interactions with normal B cells may be a key contributor to clinical CAR T cell responses.

PD1 Expression in EGFRvIII-Directed CAR T Cell Infusion Product for Glioblastoma Is Associated with Clinical Response.

The epidermal growth factor receptor variant III (EGFRvIII) has been investigated as a therapeutic target for chimeric antigen receptor (CAR) T cell therapy in glioblastoma. Earlier research demonstrated that phenotypic and genotypic characteristics in T cells and CAR T product predicted therapeutic success in hematologic malignancies, to date no determinants for clinical response in solid tumors have been identified. We analyzed apheresis and infusion products from the first-in-human trial of EGFRvIII-directed CAR T for recurrent glioblastoma (NCT02209376) by flow cytometry. Clinical response was quantified via engraftment in peripheral circulation and progression-free survival (PFS), as determined by the time from CAR T infusion to first radiographic evidence of progression. The CD4+CAR T cell population in patient infusion products demonstrated PD1 expression which positively correlated with AUC engraftment and PFS. On immune checkpoint inhibitor analysis, CTLA-4, TIM3, and LAG3 did not exhibit significant associations with engraftment or PFS. The frequencies of PD1+GZMB+ and PD1+HLA-DR+ CAR T cells in the CD4+ infusion products were directly proportional to AUC and PFS. No significant associations were observed within the apheresis products. In summary, PD1 in CAR T infusion products predicted peripheral engraftment and PFS in recurrent glioblastoma.

Temperature Trajectory Sub-phenotypes and the Immuno-Inflammatory Response in Pediatric Sepsis.

OBJECTIVE: Heterogeneity has hampered sepsis trials, and sub-phenotyping may assist with enrichment strategies. However, biomarker-based strategies are difficult to operationalize. Four sub-phenotypes defined by distinct temperature trajectories in the first 72 h have been reported in adult sepsis. Given the distinct epidemiology of pediatric sepsis, the existence and relevance of temperature trajectory-defined sub-phenotypes in children is unknown. We aimed to classify septic children into de novo sub-phenotypes derived from temperature trajectories in the first 72 h, and compare cytokine, immune function, and immunometabolic markers across subgroups. METHODS: This was a secondary analysis of a prospective cohort of 191 critically ill septic children recruited from a single academic pediatric intensive care unit. We performed group-based trajectory modeling using temperatures over the first 72 h of sepsis to identify latent profiles. We then used mixed effects regression to determine if temperature trajectory-defined sub-phenotypes were associated with cytokine levels, immune function, and mitochondrial respiration. RESULTS: We identified four temperature trajectory-defined sub-phenotypes: hypothermic, normothermic, hyperthermic fast-resolvers, and hyperthermic slow-resolvers. Hypothermic patients were less often previously healthy and exhibited lower levels of pro- and anti-inflammatory cytokines and chemokines. Hospital mortality did not differ between hypothermic children (17%) and other sub-phenotypes (3-11%; P = 0.26). CONCLUSIONS: Critically ill septic children can be categorized into temperature trajectory-defined sub-phenotypes that parallel adult sepsis. Hypothermic children exhibit a blunted cytokine and chemokine profile. Group-based trajectory modeling has utility for identifying subtypes of clinical syndromes by incorporating readily available longitudinal data, rather than relying on inputs from a single timepoint.

Tumor vaccines and beyond.

For the last two decades the immunotherapy of patients with solid and hematopoietic tumors has met with variable success. We have reviewed the field of tumor vaccines to examine what has worked and what has not, why this has been the case, how the anti-tumor responses were examined, and how we can make tumor immunity successful for the majority of individuals rather than for the exceptional patients who currently show successful immune responses against their tumors.

Improving and Maintaining Responses in Pediatric B-Cell Acute Lymphoblastic Leukemia Chimeric Antigen Receptor-T Cell Therapy.

ABSTRACT: Chimeric antigen receptor T therapy has heralded a new era in the treatment of acute lymphoblastic leukemia (ALL) and other hematologic malignancies. In this autologous immunotherapy, patient-derived T cells are genetically engineered and then infused back to kill the leukemia cells. The observed response rates in ALL are a testament to the success of this therapy. However, there have been instances where the patients either did not respond or relapsed after initial response. Emergence of resistance due to antigen loss and T-cell exhaustion has been observed. This poses a challenge in making this therapy successful for every ALL patient and warrants deeper understanding of emergence of resistance and potential approaches to overcome them. Here we discuss current perspectives and advances in this area.

Case Report: Prolonged Survival Following EGFRvIII CAR T Cell Treatment for Recurrent Glioblastoma.

Autologous chimeric antigen receptor (CAR) T cells targeted to epidermal growth factor receptor variant III (CAR T-EGFRvIII) have been developed and administered experimentally to treat patients with IDH1 wildtype recurrent glioblastoma (rGBM) (NCT02209376). We report the case of a 59-year-old patient who received a single peripheral infusion of CAR T-EGFRvIII cells and survived 36 months after disease recurrence, exceeding expected survival for recurrent glioblastoma. Post-infusion histopathologic analysis of tissue obtained during a second stage surgical resection revealed immunosuppressive adaptive changes in the tumor tissue as well as reduced EGFRvIII expression. Serial brain imaging demonstrated a significant reduction in relative cerebral blood volume (rCBV), a measure strongly associated with tumor proliferative activity, at early time points following CAR T treatment. Notably, CAR T-EGFRvIII cells persisted in her peripheral circulation during 29 months of follow-up, the longest period of CAR T persistence reported in GBM trials to date. These findings in a long-term survivor show that peripherally administered CAR T-EGFRvIII cells can persist for years in the circulation and suggest that this cell therapy approach could be optimized to achieve broader efficacy in recurrent GBM patients.

Cancer stem cells: advances in biology and clinical translation-a Keystone Symposia report.

The test for the cancer stem cell (CSC) hypothesis is to find a target expressed on all, and only CSCs in a patient tumor, then eliminate all cells with that target that eliminates the cancer. That test has not yet been achieved, but CSC diagnostics and targets found on CSCs and some other cells have resulted in a few clinically relevant therapies. However, it has become apparent that eliminating the subset of tumor cells characterized by self-renewal properties is essential for long-term tumor control. CSCs are able to regenerate and initiate tumor growth, recapitulating the heterogeneity present in the tumor before treatment. As great progress has been made in identifying and elucidating the biology of CSCs as well as their interactions with the tumor microenvironment, the time seems ripe for novel therapeutic strategies that target CSCs to find clinical applicability. On May 19-21, 2021, researchers in cancer stem cells met virtually for the Keystone eSymposium "Cancer Stem Cells: Advances in Biology and Clinical Translation" to discuss recent advances in the understanding of CSCs as well as clinical efforts to target these populations.