Low booster uptake in cancer patients despite health benefits.

Patients with cancer are at increased risk of death from COVID-19 and have reduced immune responses to SARS-CoV2 vaccines, necessitating regular boosters. We performed comprehensive chart reviews, surveys of patients attitudes, serology for SARS-CoV-2 antibodies and T-cell receptor (TCR) ß sequencing for cellular responses on a cohort of 982 cancer patients receiving active cancer therapy accrued between November-3-2020 and Mar-31-2023. We found that 92·3% of patients received the primer vaccine, 70·8% received one monovalent booster, but only 30·1% received a bivalent booster. Booster uptake was lower under age 50, and among African American or Hispanic patients. Nearly all patients seroconverted after 2+ booster vaccinations (>99%) and improved cellular responses, demonstrating that repeated boosters could overcome poor response to vaccination. Receipt of booster vaccinations was associated with a lower risk of all-cause mortality (HR=0·61, P=0·024). Booster uptake in high-risk cancer patients remains low and strategies to encourage booster uptake are needed. Highlights: COVID-19 booster vaccinations increase antibody levels and maintain T-cell responses against SARS-CoV-2 in patients receiving various anti-cancer therapiesBooster vaccinations reduced all-cause mortality in patientsA significant proportion of patients remain unboosted and strategies are needed to encourage patients to be up-to-date with vaccinations.

Deciphering intratumoral heterogeneity using integrated clonal tracking and single-cell transcriptome analyses.

Cellular heterogeneity is a major cause of treatment resistance in cancer. Despite recent advances in single-cell genomic and transcriptomic sequencing, it remains difficult to relate measured molecular profiles to the cellular activities underlying cancer. Here, we present an integrated experimental system that connects single cell gene expression to heterogeneous cancer cell growth, metastasis, and treatment response. Our system integrates single cell transcriptome profiling with DNA barcode based clonal tracking in patient-derived xenograft models. We show that leukemia cells exhibiting unique gene expression respond to different chemotherapies in distinct but consistent manners across multiple mice. In addition, we uncover a form of leukemia expansion that is spatially confined to the bone marrow of single anatomical sites and driven by cells with distinct gene expression. Our integrated experimental system can interrogate the molecular and cellular basis of the intratumoral heterogeneity underlying disease progression and treatment resistance.

Efficacy of inotuzumab ozogamicin in patients with Philadelphia chromosome-positive relapsed/refractory acute lymphoblastic leukemia.

BACKGROUND: Patients with relapsed/refractory (R/R) Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) have a poor prognosis and limited treatment options. METHODS: The efficacy of inotuzumab ozogamicin (InO), a humanized anti-CD22 monoclonal antibody conjugated to the cytotoxic antibiotic calicheamicin, was evaluated in R/R ALL patients in the phase 1/2 study 1010 (NCT01363297) and open-label, randomized, phase 3 study 1022 (INO-VATE; NCT01564784). This analysis focused specifically on Ph+ R/R ALL patients. In study 1022, Ph+ patients were randomly assigned 1:1 to InO (n = 22) or standard intensive chemotherapy (SC) (n = 27) and 16 Ph+ patients in study 1010 received InO. RESULTS: In study 1022, rates of complete remission/complete remission with incomplete hematologic recovery (CR/CRi) and minimal residual disease (MRD) negativity (patients achieving CR/CRi) were higher with InO (CR/CRi = 73%; MRD = 81%) versus SC (CR/CRi = 56%; MRD = 33%). The corresponding rates in study 1010 were 56% (CR/CRi) and 100% (MRD). The hematopoietic stem cell transplantation (HSCT) rate in study 1022 was 41% versus 19% for InO versus SC; however, there was no benefit in overall survival (median OS: 8.7 vs 8.4 months; hazard ratio, 1.17 [95% CI, 0.64-2.14]). The probability of being event-free (progression-free survival) at 12 months was greater with InO versus SC (20.1% vs 4.8%). CONCLUSION: Given the substantial improvement in responses and rates of HSCT, InO is an important treatment option for patients with R/R Ph+ ALL. Future studies need to consider better characterization of disease characteristics, more sensitive MRD measurements, MRD-directed therapy before HSCT, and potentially combination therapies, including tyrosine kinase inhibitors.

Public Concern About Monitoring Twitter Users and Their Conversations to Recruit for Clinical Trials: Survey Study.

BACKGROUND: Social networks such as Twitter offer the clinical research community a novel opportunity for engaging potential study participants based on user activity data. However, the availability of public social media data has led to new ethical challenges about respecting user privacy and the appropriateness of monitoring social media for clinical trial recruitment. Researchers have voiced the need for involving users' perspectives in the development of ethical norms and regulations. OBJECTIVE: This study examined the attitudes and level of concern among Twitter users and nonusers about using Twitter for monitoring social media users and their conversations to recruit potential clinical trial participants. METHODS: We used two online methods for recruiting study participants: the open survey was (1) advertised on Twitter between May 23 and June 8, 2017, and (2) deployed on TurkPrime, a crowdsourcing data acquisition platform, between May 23 and June 8, 2017. Eligible participants were adults, 18 years of age or older, who lived in the United States. People with and without Twitter accounts were included in the study. RESULTS: While nearly half the respondents-on Twitter (94/603, 15.6%) and on TurkPrime (509/603, 84.4%)-indicated agreement that social media monitoring constitutes a form of eavesdropping that invades their privacy, over one-third disagreed and nearly 1 in 5 had no opinion. A chi-square test revealed a positive relationship between respondents' general privacy concern and their average concern about Internet research (P<.005). We found associations between respondents' Twitter literacy and their concerns about the ability for researchers to monitor their Twitter activity for clinical trial recruitment (P=.001) and whether they consider Twitter monitoring for clinical trial recruitment as eavesdropping (P<.001) and an invasion of privacy (P=.003). As Twitter literacy increased, so did people's concerns about researchers monitoring Twitter activity. Our data support the previously suggested use of the nonexceptionalist methodology for assessing social media in research, insofar as social media-based recruitment does not need to be considered exceptional and, for most, it is considered preferable to traditional in-person interventions at physical clinics. The expressed attitudes were highly contextual, depending on factors such as the type of disease or health topic (eg, HIV/AIDS vs obesity vs smoking), the entity or person monitoring users on Twitter, and the monitored information. CONCLUSIONS: The data and findings from this study contribute to the critical dialogue with the public about the use of social media in clinical research. The findings suggest that most users do not think that monitoring Twitter for clinical trial recruitment constitutes inappropriate surveillance or a violation of privacy. However, researchers should remain mindful that some participants might find social media monitoring problematic when connected with certain conditions or health topics. Further research should isolate factors that influence the level of concern among social media users across platforms and populations and inform the development of more clear and consistent guidelines.

Multiplex protein detection on circulating tumor cells from liquid biopsies using imaging mass cytometry.

Molecular analysis of circulating and disseminated tumor cells (CTCs/DTCs) has great potential as a means for continuous evaluation of prognosis and treatment efficacy in near-real time through minimally invasive liquid biopsies. To realize this potential, however, methods for molecular analysis of these rare cells must be developed and validated. Here, we describe the integration of imaging mass cytometry (IMC) using metal-labeled antibodies as implemented on the Fluidigm Hyperion Imaging System into the workflow of the previously established High Definition Single Cell Analysis (HD-SCA) assay for liquid biopsies, along with methods for image analysis and signal normalization. Using liquid biopsies from a metastatic prostate cancer case, we demonstrate that IMC can extend the reach of CTC characterization to include dozens of protein biomarkers, with the potential to understand a range of biological properties that could affect therapeutic response, metastasis and immune surveillance when coupled with simultaneous phenotyping of thousands of leukocytes.

Hepatic adverse event profile of inotuzumab ozogamicin in adult patients with relapsed or refractory acute lymphoblastic leukaemia: results from the open-label, randomised, phase 3 INO-VATE study.

BACKGROUND: The INO-VATE study demonstrated efficacy and safety of inotuzumab ozogamicin versus standard care in adults with relapsed or refractory B-cell acute lymphoblastic leukaemia. Here, we report the frequency of, and potential risk factors for, hepatotoxicity in patients in this trial and after treatment and subsequent haemopoietic stem-cell transplantation (HSCT). METHODS: In this open-label, phase 3, multicentre, international study, adults with relapsed or refractory, CD22-positive, Philadelphia chromosome (Ph)-positive or Ph-negative B-cell acute lymphoblastic leukaemia who were due to receive first or second salvage treatment were randomly assigned (1:1) via an interactive voice response system to receive inotuzumab ozogamicin (starting dose 1·8 mg/m2 per cycle [0·8 mg/m2 on day 1; 0·5 mg/m2 on days 8 and 15 of a 21-28 day cycle for ≤6 cycles]) or standard care (either fludarabine plus cytarabine plus granulocyte colony-stimulating factor, mitoxantrone plus cytarabine, or high-dose cytarabine). Stratification factors at randomisation were duration of first remission (<12 months vs ≥12 months), salvage treatment phase (first vs second), and age (<55 years vs ≥55 years). We present data up to March 8, 2016. At this cutoff date, all patients had been discontinued from treatment but 54 patients were continuing in long-term follow-up. Long-term follow-up has now been completed, with the final patient's last visit on Jan 4, 2017. This prespecified safety analysis describes investigator-assessed treatment-emergent hepatotoxicity, including sinusoidal obstruction syndrome (also known as veno-occlusive disease) in patients during study treatment or thereafter (without follow-up HSCT) and after study treatment and subsequent HSCT, for all patients who received at least one dose of study treatment. This study is registered with ClinicalTrials.gov, number NCT01564784. FINDINGS: Between Aug 27, 2012, and and the data cutoff of March 8, 2016, 326 patients were randomly assigned to receive inotuzumab ozogamicin (n=164) or standard care (n=162). 164 patients in the inotuzumab ozogamicin group and 143 in the standard care group received at least one dose of study treatment and were included in the safety population. At data cutoff, median duration of treatment (induction) was 8·9 weeks (IQR 4·1-13·1) in the inotuzumab ozogamicin group and 0·9 weeks (0·9-1·1) in the standard care group. Treatment-emergent hepatotoxicities (of all grades) were more frequent in the inotuzumab ozogamicin group (83 [51%] of 164 patients) than in the standard care group (49 [34%] of 143 patients). The frequency of sinusoidal obstruction syndrome-comprising events occurring during treatment (or follow-up without HSCT) and after treatment and subsequent HSCT-was higher in the inotuzumab ozogamicin group (22 [13%]; 18 [82%] of which were grade 3 or worse) than in the standard care group (one [<1%]). During study therapy or follow-up without HSCT, five (3%) patients in the inotuzumab ozogamicin group developed sinusoidal obstruction syndrome compared with no patients in the standard care group. Of the 77 patients who received inotuzumab ozogamicin and proceeded to HSCT, 17 (22%) had sinusoidal obstruction syndrome; five events after follow-up HSCT were fatal. Of 32 patients who received standard care and proceeded to HSCT, one (3%) had (non-fatal) sinusoidal obstruction syndrome that was ongoing at the time of death due to septic shock. In multivariate analysis, conditioning with two alkylating agents (p=0·015 vs one alkylating agent) and last available pre-HSCT bilirubin concentration of greater than or equal to the upper limit of normal (ULN; p=0·009 vs

GLI3 repressor determines Hedgehog pathway activation and is required for response to SMO antagonist glasdegib in AML.

The Hedgehog (Hh) signaling pathway is activated in many cancers and is a promising target for therapeutic development. Deletions in the receptor Patched (PTCH) or activating mutations in Smoothened (SMO) have been reported in basal cell carcinoma and medulloblastoma, but are largely absent in most tumor types. Therefore, the mechanism of pathway activation in most cancers, including hematological malignancies, remains unknown. In normal tissues, Hh pathway activation via PTCH/SMO causes an increase in the downstream transcriptional activator GLI1 and a decrease in the GLI3 transcriptional repressor (GLI3R). In this article, we confirm that the Hh pathway is active in acute myeloid leukemia (AML), however, this activity is largely independent of SMO. Epigenetic and gene expression analysis of The Cancer Genome Atlas AML data set reveals that GLI3 expression is silenced in most AML patient samples, and the GLI3 locus is abnormally methylated. We show that GLI3R is required for the therapeutic effect of SMO antagonists in AML samples and restoration of GLI3R suppresses the growth of AML. We additionally demonstrate that GLI3R represses AML growth by downregulating AKT expression. In summary, this study provides the first evidence that GLI3R plays an essential role in SMO-independent Hh signaling in AML, and suggests that GLI3R could serve as a potential biomarker for patient selection in SMO antagonist clinical trials. Furthermore, these data support rational combinations of hypomethylating agents with SMO antagonists in clinical trials.

EPHB4 is a therapeutic target in AML and promotes leukemia cell survival via AKT.

EPHB4, an ephrin type B receptor, is implicated in the growth of several epithelial tumors and is a promising target in cancer therapy; however, little is known about its role in hematologic malignancies. In this article, we show that EPHB4 is highly expressed in ∼30% of acute myeloid leukemia (AML) samples. In an unbiased RNA interference screen of primary leukemia samples, we found that EPHB4 drives survival in a subset of AML cases. Knockdown of EPHB4 inhibits phosphatidylinositol 3-kinase/AKT signaling, and this is accompanied by a reduction in cell viability, which can be rescued by a constitutively active form of AKT. Finally, targeting EPHB4 with a highly specific monoclonal antibody (MAb131) is effective against AML in vitro and in vivo. EPHB4 is therefore a potential target in AML with high EPHB4 expression.

Primary cilia are present on human blood and bone marrow cells and mediate Hedgehog signaling.

Primary cilia are nonmotile, microtubule-based organelles that are present on the cellular membrane of all eukaryotic cells. Functional cilia are required for the response to developmental signaling pathways such as Hedgehog (Hh) and Wnt/ß-catenin. Although the Hh pathway has been shown to be active in leukemia and other blood cancers, there have been no reports describing the presence of primary cilia in human blood or leukemia cells. In the present study, we show that nearly all human blood and bone marrow cells have primary cilia (97-99%). In contrast, primary cilia on AML cell lines (KG1, KG1a, and K562) were less frequent (10-36% of cells) and were often shorter and dysmorphic, with less well-defined basal bodies. Finally, we show that treatment of blood cells with the Hh pathway ligand Sonic Hedgehog (SHh) causes translocation of Smoothened (SMO) to the primary cilia and activation of Hh target genes, demonstrating that primary cilia in blood cells are functional and participate in Hh signaling. Loss of primary cilia on leukemia cells may have important implications for aberrant pathway activation and response to SMO inhibitors currently in clinical development.

Integration of Hedgehog and mutant FLT3 signaling in myeloid leukemia.

FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutations resulting in constitutive kinase activity are common in acute myeloid leukemia (AML) and carry a poor prognosis. Several agents targeting FLT3 have been developed, but their limited clinical activity suggests that the inhibition of other factors contributing to the malignant phenotype is required. We examined gene expression data sets as well as primary specimens and found that the expression of GLI2, a major effector of the Hedgehog (Hh) signaling pathway, was increased in FLT3-ITD compared to wild-type FLT3 AML. To examine the functional role of the Hh pathway, we studied mice in which Flt3-ITD expression results in an indolent myeloproliferative state and found that constitutive Hh signaling accelerated the development of AML by enhancing signal transducer and activator of transcription 5 (STAT5) signaling and the proliferation of bone marrow myeloid progenitors. Furthermore, combined FLT3 and Hh pathway inhibition limited leukemic growth in vitro and in vivo, and this approach may serve as a therapeutic strategy for FLT3-ITD AML.

Chloroquine improves survival and hematopoietic recovery after lethal low-dose-rate radiation.

PURPOSE: We have previously shown that the antimalarial agent chloroquine can abrogate the lethal cellular effects of low-dose-rate (LDR) radiation in vitro, most likely by activating the ataxia-telangiectasia mutated (ATM) protein. Here, we demonstrate that chloroquine treatment also protects against lethal doses of LDR radiation in vivo. METHODS AND MATERIALS: C57BL/6 mice were irradiated with a total of 12.8 Gy delivered at 9.4 cGy/hour. ATM null mice from the same background were used to determine the influence of ATM. Chloroquine was administered by two intraperitoneal injections of 59.4 µg per 17 g of body weight, 24 hours and 4 hours before irradiation. Bone marrow cells isolated from tibia, fibula, and vertebral bones were transplanted into lethally irradiated CD45 congenic recipient mice by retroorbital injection. Chimerism was assessed by flow cytometry. In vitro methylcellulose colony-forming assay of whole bone marrow cells and fluorescence activated cell sorting analysis of lineage depleted cells were used to assess the effect of chloroquine on progenitor cells. RESULTS: Mice pretreated with chloroquine before radiation exhibited a significantly higher survival rate than did mice treated with radiation alone (80% vs. 31%, p = 0.0026). Chloroquine administration before radiation did not affect the survival of ATM null mice (p = 0.86). Chloroquine also had a significant effect on the early engraftment of bone marrow cells from the irradiated donor mice 6 weeks after transplantation (4.2% vs. 0.4%, p = 0.015). CONCLUSION: Chloroquine administration before radiation had a significant effect on the survival of normal but not ATM null mice, strongly suggesting that the in vivo effect, like the in vitro effect, is also ATM dependent. Chloroquine improved the early engraftment of bone marrow cells from LDR-irradiated mice, presumably by protecting the progenitor cells from radiation injury. Chloroquine thus could serve as a very useful drug for protection against the harmful effects of LDR radiation.

Not so benign haematology: anaemia of the elderly.

Developed countries, such as the United Kingdom, are experiencing a change in demographics resulting in the largest proportion of adults over 65 years of age that our health systems have ever experienced. As such, haematologists must be prepared to evaluate and treat anaemia in a more complicated patient population, but sufficient evidence-based guidelines are lacking. Critical next steps that must be taken to ensure the best care of this population include the determination of appropriate haemoglobin concentrations for older adults in light of age, gender, race, and comorbidities; the development of interventional trials that address physical performance outcomes in addition to haemoglobin targets; and translational studies which address the molecular pathogenesis of anaemia in older adults with the most advanced scientific approaches.

The redox-sensitive transcription factor Nrf2 regulates murine hematopoietic stem cell survival independently of ROS levels.

Several studies have found that high levels of reactive oxidative species (ROS) are associated with stem cell dysfunction. In the present study, we investigated the role of nuclear factor erythroid-2-related factor 2 (Nrf2), a master regulator of the antioxidant response, and found that it is required for hematopoietic stem progenitor cell (HSPC) survival and myeloid development. Although the loss of Nrf2 leads to increased ROS in most tissues, basal ROS levels in Nrf2-deficient (Nrf2(-/-)) BM were not elevated compared with wild-type. Nrf2(-/-) HSPCs, however, had increased rates of spontaneous apoptosis and showed decreased survival when exposed to oxidative stress. Nrf2(-/-) BM demonstrated defective stem cell function, as evidenced by reduced chimerism after transplantation that was not rescued by treatment with the antioxidant N-acetyl cysteine. Gene-expression profiling revealed that the levels of prosurvival cytokines were reduced in Nrf2(-/-) HSPCs. Treatment with the cytokine G-CSF improved HSPC survival after exposure to oxidative stress and rescued the transplantation defect in Nrf2(-/-) cells despite increases in ROS induced by cytokine signaling. These findings demonstrate a critical role for Nrf2 in hematopoiesis and stem cell survival that is independent of ROS levels.

Mantle cell lymphoma activation enhances bortezomib sensitivity.

Patients with mantle cell lymphoma (MCL) typically respond to initial treatment but subsequently relapse. This pattern suggests that a population of MCL cells is both drug resistant and capable of clonogenic growth. The intracellular enzyme retinaldehyde dehydrogenase (ALDH) provides resistance to several toxic agents. ALDH can also identify stem cells in normal adult tissues and tumorigenic cancer stem cells in several human malignancies. We studied ALDH expression in MCL and found small populations of ALDH(+) cells that were highly clonogenic. Moreover, ALDH(+) MCL cells were relatively quiescent and resistant to a wide range of agents. Normal B cells can be activated by specific unmethylated cytosine-phosphate-guanosine (CpG) DNA motifs through toll-like receptor 9, and we found that the synthetic CpG oligonucleotide 2006 (CpG) reduced the frequency of quiescent ALDH(+) MCL cells, induced terminal plasma cell differentiation, and limited tumor formation in vitro and in vivo. Treatment with CpG also significantly enhanced the activity of the proteasome inhibitor bortezomib that was associated with induction of the unfolded protein response. Our data suggest that CpG may target clonogenic and resistant ALDH(+) cells as well as improve the activity of proteasome inhibitors in MCL.

Targeting Hedgehog--a cancer stem cell pathway.

The Hedgehog (Hh) pathway has been implicated in a wide variety of human tumors, and early clinical trials with pathway antagonists have validated Hh signaling as a bona fide anticancer target. Despite these encouraging results, several issues surrounding the basic biology of the Hh pathway in human cancers remain unclear. These include the influence of specific oncogenic events on Hh signal transduction, the precise mode of Hh signaling (i.e., autocrine or paracrine) that occurs within human tumors, and the best means to inhibit aberrant pathway activity in the clinical setting. The cancer stem cell (CSC) hypothesis may explain a number of clinical phenomena, such as unchecked self-renewal and the development of metastatic disease, and to some extent, the Hh signaling pathway has been implicated in all of these processes. Therefore, Hh pathway inhibitors may also represent some of the first agents to formally examine the CSC hypothesis in the clinical setting. The diverse nature of Hh signaling in human cancers suggests that disease-specific factors must be carefully considered to identify the optimal use of novel pathway inhibitors.

Self-renewal of acute lymphocytic leukemia cells is limited by the Hedgehog pathway inhibitors cyclopamine and IPI-926.

Conserved embryonic signaling pathways such as Hedgehog (Hh), Wingless and Notch have been implicated in the pathogenesis of several malignancies. Recent data suggests that Hh signaling plays a role in normal B-cell development, and we hypothesized that Hh signaling may be important in precursor B-cell acute lymphocytic leukemia (B-ALL). We found that the expression of Hh pathway components was common in human B-ALL cell lines and clinical samples. Moreover, pathway activity could be modulated by Hh ligand or several pathway inhibitors including cyclopamine and the novel SMOOTHENED (SMO) inhibitor IPI-926. The inhibition of pathway activity primarily impacted highly clonogenic B-ALL cells expressing aldehyde dehydrogenase (ALDH) by limiting their self-renewal potential both in vitro and in vivo. These data demonstrate that Hh pathway activation is common in B-ALL and represents a novel therapeutic target regulating self-renewal and persistence of the malignant clone.

Smoothening the controversial role of hedgehog in hematopoiesis.

The role of multiple developmental signaling pathways in the regulation of hematopoiesis has been controversial. In this issue of Cell Stem Cell, two separate reports (Hofmann et al., 2009; Gao et al., 2009) examine whether the Hedgehog signaling pathway modulates normal adult hematopoietic stem cells and blood formation.

Molecular profiling of hematopoietic stem cells.

Gene expression profiling using microarrays is a powerful method for studying the biology of hematopoietic stem cells (HSCs). Here, we present methods for activating HSCs with the chemotherapeutic drug 5-Fluorouracil, isolating HSCs from whole bone marrow, and performing microarray analysis. We also discuss quality control criteria for identifying good arrays and bioinformatics strategies for analyzing them. Using these methods, we have characterized the gene expression signatures of HSC quiescence and proliferation and have constructed a molecular model of HSC activation and self-renewal.

Hematopoietic fingerprints: an expression database of stem cells and their progeny.

Hematopoietic stem cells (HSCs) continuously regenerate the hematologic system, yet few genes regulating this process have been defined. To identify candidate factors involved in differentiation and self-renewal, we have generated an expression database of hematopoietic stem cells and their differentiated progeny, including erythrocytes, granulocytes, monocytes, NK cells, activated and naive T cells, and B cells. Bioinformatic analysis revealed HSCs were more transcriptionally active than their progeny and shared a common activation mechanism with T cells. Each cell type also displayed unique biases in the regulation of particular genetic pathways, with Wnt signaling particularly enhanced in HSCs. We identified approximately 100-400 genes uniquely expressed in each cell type, termed lineage "fingerprints." In overexpression studies, two of these genes, Zfp 105 from the NK cell lineage, and Ets2 from the monocyte lineage, were able to significantly influence differentiation toward their respective lineages, demonstrating the utility of the fingerprints for identifying genes that regulate differentiation.

Evidence for diversity in transcriptional profiles of single hematopoietic stem cells.

Hematopoietic stem cells replenish all the cells of the blood throughout the lifetime of an animal. Although thousands of stem cells reside in the bone marrow, only a few contribute to blood production at any given time. Nothing is known about the differences between individual stem cells that dictate their particular state of activation readiness. To examine such differences between individual stem cells, we determined the global gene expression profile of 12 single stem cells using microarrays. We showed that at least half of the genetic expression variability between 12 single cells profiled was due to biological variation in 44% of the genes analyzed. We also identified specific genes with high biological variance that are candidates for influencing the state of readiness of individual hematopoietic stem cells, and confirmed the variability of a subset of these genes using single-cell real-time PCR. Because apparent variation of some genes is likely due to technical factors, we estimated the degree of biological versus technical variation for each gene using identical RNA samples containing an RNA amount equivalent to that of single cells. This enabled us to identify a large cohort of genes with low technical variability whose expression can be reliably measured on the arrays at the single-cell level. These data have established that gene expression of individual stem cells varies widely, despite extremely high phenotypic homogeneity. Some of this variation is in key regulators of stem cell activity, which could account for the differential responses of particular stem cells to exogenous stimuli. The capacity to accurately interrogate individual cells for global gene expression will facilitate a systems approach to biological processes at a single-cell level.