IMPORTANCEHospitalized patients with COVID-19 are at risk for venous and arterial thromboembolism and death. Optimal thromboprophylaxis dosing in high-risk patients is unknown.OBJECTIVE To evaluate the effects of therapeutic-dose low-molecular-weight heparin (LMWH) vs institutional standard prophylactic or intermediate-dose heparins for thromboprophylaxis in high-risk hospitalized patients with COVID-19. DESIGN, SETTING, AND PARTICIPANTSThe HEP-COVID multicenter randomized clinical trial recruited hospitalized adult patients with COVID-19 with D-dimer levels more than 4 times the upper limit of normal or sepsis-induced coagulopathy score of 4 or greater from May 8, 2020, through May 14, 2021, at 12 academic centers in the US.INTERVENTIONS Patients were randomized to institutional standard prophylactic or intermediate-dose LMWH or unfractionated heparin vs therapeutic-dose enoxaparin, 1 mg/kg subcutaneous, twice daily if creatinine clearance was 30 mL/min/1.73 m 2 or greater (0.5 mg/kg twice daily if creatinine clearance was 15-29 mL/min/1.73 m 2 ) throughout hospitalization. Patients were stratified at the time of randomization based on intensive care unit (ICU) or non-ICU status. MAIN OUTCOMES AND MEASURESThe primary efficacy outcome was venous thromboembolism (VTE), arterial thromboembolism (ATE), or death from any cause, and the principal safety outcome was major bleeding at 30 ± 2 days. Data were collected and adjudicated locally by blinded investigators via imaging, laboratory, and health record data. RESULTSOf 257 patients randomized, 253 were included in the analysis (mean [SD] age, 66.7 [14.0] years; men, 136 [53.8%]; women, 117 [46.2%]); 249 patients (98.4%) met inclusion criteria based on D-dimer elevation and 83 patients (32.8%) were stratified as ICU-level care. There were 124 patients (49%) in the standard-dose vs 129 patients (51%) in the therapeutic-dose group. The primary efficacy outcome was met in 52 of 124 patients (41.9%) (28.2% VTE, 3.2% ATE, 25.0% death) with standard-dose heparins vs 37 of 129 patients (28.7%) (11.7% VTE, 3.2% ATE, 19.4% death) with therapeutic-dose LMWH (relative risk [RR], 0.68; 95% CI, 0.49-0.96; P = .03), including a reduction in thromboembolism (29.0% vs 10.9%; RR, 0.37; 95% CI, 0.21-0.66; P < .001). The incidence of major bleeding was 1.6% with standard-dose vs 4.7% with therapeutic-dose heparins (RR, 2.88; 95% CI, 0.59-14.02; P = .17). The primary efficacy outcome was reduced in non-ICU patients (36.1% vs 16.7%; RR, 0.46; 95% CI, 0.27-0.81; P = .004) but not ICU patients (55.3% vs 51.1%; RR, 0.92; 95% CI, 0.62-1.39; P = .71). CONCLUSIONS AND RELEVANCEIn this randomized clinical trial, therapeutic-dose LMWH reduced major thromboembolism and death compared with institutional standard heparin thromboprophylaxis among inpatients with COVID-19 with very elevated D-dimer levels. The treatment effect was not seen in ICU patients.

The failure of chemotherapeutic regimens to eradicate cancers often results from the outgrowth of minor subclones with more dangerous genomic abnormalities or with self-renewing capacity. To explore such intratumor complexities in B-cell chronic lymphocytic leukemia (CLL), we measured B-cell kinetics in vivo by quantifying deuterium ( 2 H)-labeled cells as an indicator of a cell that had divided. Separating CLL clones on the basis of reciprocal densities of chemokine (C-X-C motif) receptor 4 (CXCR4) and cluster designation 5 (CD5) revealed that the CXCR4 dim CD5 bright (proliferative) fraction contained more 2 H-labeled DNA and hence divided cells than the CXCR4 bright CD5 dim (resting) fraction. This enrichment was confirmed by the relative expression of two cell cycle-associated molecules in the same fractions, Ki-67 and minichromosome maintenance protein 6 (MCM6). Comparisons of global gene expression between the CXCR4 dim CD5 bright and CXCR4 bright CD5 dim fractions indicated higher levels of pro-proliferation and antiapoptotic genes and genes involved in oxidative injury in the proliferative fraction. An extended immunophenotype was also defined, providing a wider range of surface molecules characteristic of each fraction. These intraclonal analyses suggest a model of CLL cell biology in which the leukemic clone contains a spectrum of cells from the proliferative fraction, enriched in recently divided robust cells that are lymphoid tissue emigrants, to the resting fraction enriched in older, less vital cells that need to immigrate to lymphoid tissue or die. The model also suggests several targets preferentially expressed in the two populations amenable for therapeutic attack. Finally, the study lays the groundwork for future analyses that might provide a more robust understanding of the development and clonal evolution of this currently incurable disease.

IntroductionB-cell chronic lymphocytic leukemia (B-CLL) is a clonal disease of an apparently slowly dividing B lymphocyte that expresses a distinct cell surface phenotype (CD19 ϩ , CD5 ϩ , CD23 ϩ cells with diminished surface membrane immunoglobulin). 1 Subgroups of B-CLL exist, defined by differences in lymphocyte doubling times in vivo, 2 variable degrees of somatic mutation of the immunoglobulin V (Ig V)-region genes in the leukemic cells, 3,4 and expression of cell surface 5 and intracellular 6,7 proteins. Each of these subgroups is accompanied by significant differences in clinical course and outcome. 2,[6][7][8][9] Membership in one of these subgroups may reflect differences in the antigenic experiences of the precursor B cells (naive vs memory), the manner in which these precursor cells were triggered (T-cell dependently or T-cell independently), or the level of spontaneous or induced cell cycling. 10 Defining the relative importance of these issues may provide clues to the development and evolution of B-CLL.Telomere lengths can serve as indicators of the replicative history of individual cells because these chromosomal segments shorten with each cell division. 11 As a consequence of the inability to adequately maintain telomere length, a slow but inexorable decrease in a cell's replicative potential accompanies cellular division, 12,13 and is also part of the normal aging process. 14-16 Two processes counteract telomere shortening and restore telomeric regions in normal and transformed cells: the activity of the enzyme telomerase [17][18][19][20] and an alternative lengthening of telomere (ALT) pathway. 21,22 Of special note is the observation that during a classical T-cell-mediated germinal center (GC) reaction, normal B cells produce high levels of telomerase and exhibit telomere lengthening. [23][24][25][26] This process presumably permits those B lymphocytes that develop newly diversified and advantageous B-cell receptors to maintain viability and to participate further in adaptive immune responses.As observed in many solid 27 and hematologic 18-20 cancers, B-CLL cells have short telomere lengths. 28 In this study, we analyzed telomere length as a means to infer the number of cell divisions that individual leukemic lymphocytes underwent and the relationship between telomere length and telomerase activity in the same cells. We were especially interested in trying to define differences in telomere length between the 2 Ig V gene mutationdefined B-CLL subgroups and to decipher to what degree this shortening reflects preleukemic events compared with leukemic events. We found that B-CLL cells have shorter telomeres than B cells or isolated CD5 ϩ and CD5 Ϫ B cells from a group of healthy For personal use only. on May 9, 2018. by guest www.bloodjournal.org From subjects matched for age. Moreover, we identified differences in telomere length and telomerase activity within the 2 Ig V gene mutation B-CLL subgroups that possibly indicate different replicative and telomere restorative histories for the cells and ...

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Clonal evolution and outgrowth of cellular variants with additional chromosomal abnormalities are major causes of disease progression in chronic lymphocytic leukemia (CLL). Because new DNA lesions occur during S phase, proliferating cells are at the core of this problem. In this study, we used in vivo deuterium ( 2 H) labeling of CLL cells to better understand the phenotype of proliferating cells in 13 leukemic clones. In each case, there was heterogeneity in cellular proliferation, with a higher fraction of newly produced CD38 ؉ cells compared with CD38 ؊ counterparts. On average, there were 2-fold higher percentages of newly born cells in the CD38 ؉ fraction than in CD38 ؊ cells; when analyzed on an individual patient basis, CD38 ؉ 2 H-labeled cells ranged from 6.6% to 73%. Based on distinct kinetic patterns, interclonal heterogeneity was also observed. Specifically, 4 patients exhibited a delayed appearance of newly produced CD38 ؉ cells in the blood, higher leukemic cell CXC chemokine receptor 4 (CXCR4) levels, and increased risk for lymphoid organ infiltration and poor outcome. Our data refine the proliferative compartment in CLL based on CD38 expression and suggest a relationship between in vivo kinetics, expression of a protein involved in CLL cell retention and trafficking to solid tissues, and clinical outcome. (Blood. 2009;114: 4832-4842)

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Chronic lymphocytic leukemia (CLL) cells are thought to have diminished cell-cycling capacity, a view challenged by their phenotypic resemblance to activated human B lymphocytes. The present study addresses the cell-cycling status of CLL cells, focusing on those leukemic cells expressing CD38, a molecule involved in signaling and activation that also serves as a prognostic marker in this disease. CD38(+) and CD38(-) members of individual CLL clones were analyzed for coexpression of molecules associated with cellular activation (CD27, CD62L, and CD69), cell-cycle entry (Ki-67), signaling (ZAP-70), and protection from apoptosis (telomerase and Bcl-2). Regardless of the size of the CD38(+) fraction within a CLL clone, CD38(+) subclones are markedly enriched for expression of Ki-67, ZAP-70, human telomerase reverse transcriptase, and telomerase activity. Although the percentage of cells (approximately 2%) entering the cell cycle as defined by Ki-67 expression is small, the absolute number within a clone can be sizeable and is contained primarily within the CD38(+) fraction. Despite these activation/proliferation differences, both CD38(+) and CD38(-) fractions have similar telomere lengths, suggesting that CD38 expression is dynamic and transient. These findings may help explain why high percentages of CD38(+) cells within clones are associated with poor clinical outcome.

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