Thrombosis, Bleeding, and the Observational Effect of Early Therapeutic Anticoagulation on Survival in Critically Ill Patients With COVID-19

This article is made available for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.Background: Hypercoagulability may be a key mechanism of death in patients with coronavirus disease 2019 (COVID-19). Objective: To evaluate the incidence of venous thromboembolism (VTE) and major bleeding in critically ill patients with COVID-19 and examine the observational effect of early therapeutic anticoagulation on survival. Design: In a multicenter cohort study of 3239 critically ill adults with COVID-19, the incidence of VTE and major bleeding within 14 days after intensive care unit (ICU) admission was evaluated. A target trial emulation in which patients were categorized according to receipt or no receipt of therapeutic anticoagulation in the first 2 days of ICU admission was done to examine the observational effect of early therapeutic anticoagulation on survival. A Cox model with inverse probability weighting to adjust for confounding was used. Setting: 67 hospitals in the United States. Participants: Adults with COVID-19 admitted to a participating ICU. Measurements: Time to death, censored at hospital discharge, or date of last follow-up. Results: Among the 3239 patients included, the median age was 61 years (interquartile range, 53 to 71 years), and 2088 (64.5%) were men. A total of 204 patients (6.3%) developed VTE, and 90 patients (2.8%) developed a major bleeding event. Independent predictors of VTE were male sex and higher D-dimer level on ICU admission. Among the 2809 patients included in the target trial emulation, 384 (11.9%) received early therapeutic anticoagulation. In the primary analysis, during a median follow-up of 27 days, patients who received early therapeutic anticoagulation had a similar risk for death as those who did not (hazard ratio, 1.12 [95% CI, 0.92 to 1.35]). Limitation: Observational design. Conclusion: Among critically ill adults with COVID-19, early therapeutic anticoagulation did not affect survival in the target trial emulation

Clinical and laboratory features of autoimmune hemolytic anemia associated with immune checkpoint inhibitors

Immune checkpoint inhibitors (ICPis) are a novel class of immunotherapeutic agents that have revolutionized the treatment of cancer; however, these drugs can also cause a unique spectrum of autoimmune toxicity. Autoimmune hemolytic anemia (AIHA) is a rare but often severe complication of ICPis. We identified 14 patients from 9 institutions across the US who developed ICPi-AIHA. The median interval from ICPi initiation to development of AIHA was 55 days (interquartile range [IQR], 22–110 days). Direct antiglobulin test (DAT) results were available for 13 of 14 patients: eight patients (62%) had a positive DAT and five (38%) had a negative DAT. The median pre-treatment and nadir hemoglobin concentrations were 11.8 g/dL (IQR, 10.2–12.9 g/dL) and 6.3 g/dL (IQR, 6.1–8.0 g/dL), respectively. Four patients (29%) had a pre-existing lymphoproliferative disorder, and two (14%) had a positive DAT prior to initiation of ICPi therapy. All patients were treated with glucocorticoids, with 3 requiring additional immunosuppressive therapy. Complete and partial recoveries of hemoglobin were achieved in 12 (86%) and 2 (14%) patients, respectively. Seven patients (50%) were re-challenged with ICPis, and one (14%) developed recurrent AIHA. Clinical and laboratory features of ICPi-AIHA were similar in DAT positive and negative patients. ICPi-AIHA shares many clinical features with primary AIHA; however, a unique aspect of ICPi-AIHA is a high incidence of DAT negativity. Glucocorticoids are an effective first-line treatment in the majority of patients with ICPi-AIHA, and most patients who are re-challenged with an ICPi do not appear to develop recurrence of AIHA

Clinical and laboratory features of autoimmune hemolytic anemia associated with immune checkpoint inhibitors

Immune checkpoint inhibitors (ICPis) are a novel class of immunotherapeutic agents that have revolutionized the treatment of cancer; however, these drugs can also cause a unique spectrum of autoimmune toxicity. Autoimmune hemolytic anemia (AIHA) is a rare, but often severe, complication of ICPis. We identified 14 patients from nine institutions across the United States who developed ICPi-AIHA. The median interval from ICPi initiation to development of AIHA was 55 days (interquartile range [IQR], 22-110 days). Results from the direct antiglobulin test (DAT) were available for 13 of 14 patients: 8 patients (62%) had a positive DAT and 5 (38%) had a negative DAT. The median pretreatment and nadir hemoglobin concentrations were 11.8 g/dL (IQR, 10.2-12.9 g/dL) and 6.3 g/dL (IQR, 6.1-8.0 g/dL), respectively. Four patients (29%) had a preexisting lymphoproliferative disorder, and two (14%) had a positive DAT prior to initiation of ICPi therapy. All patients were treated with glucocorticoids, with three requiring additional immunosuppressive therapy. Complete and partial recoveries of hemoglobin were achieved in 12 (86%) and 2 (14%) patients, respectively. Seven patients (50%) were rechallenged with ICPis, and one (14%) developed recurrent AIHA. Clinical and laboratory features of ICPi-AIHA were similar in DAT positive and negative patients. ICPi-AIHA shares many clinical features with primary AIHA; however, a unique aspect of ICPi-AIHA is a high incidence of DAT negativity. Glucocorticoids are an effective first-line treatment in the majority of patients with ICPi-AIHA, and most patients who are rechallenged with an ICPi do not appear to develop recurrence of AIHA

MUC1-C Inhibition Leads to Decrease in PD-L1 Levels Via up-Regulation of Micro RNAs

Acute myeloid leukemia (AML) is a lethal hematologic cancer in which the tumor microenvironment is characterized by a state of immune dysregulation that fosters disease progression. The PD-L1/PD-1 pathway confers a negative co-stimulatory signal that induces T-cell exhaustion, supports immune evasion by malignant cells and is a critical component of the immunosuppressive milieu in AML. While PD-L1 expression in AML is dynamic, little is known about the mechanisms responsible for regulating PD-L1 expression in AML. MUC1 is a heterodimeric oncoprotein aberrantly expressed in solid tumors and hematologic malignancies including AML, which supports critical aspects of the malignant phenotype including cell proliferation, self-renewal and resistance to apoptosis. Given its critical function is supporting the malignant phenotype of AML cells and its presence on leukemia stem cells, we sought to explore its role in mediating the immunosuppressive milieu of the tumor microenvironment. In the present study, we have demonstrated that suppression of MUC1-C expression via MUC1 specific shRNA or CRISPR/Cas9 gene editing results in the near abrogation of PD-L1 expression as shown by Flow Cytometry and Immuno-blotting. Interestingly, no decrease in PD-L1 mRNA expression was detected by qPCR, suggesting a post-transcriptional regulation of PD-L1 expression. Noncoding RNAs have been shown to regulate critical aspects of oncogenic phenotype through the selective binding to target mRNAs and regulation of protein translation. The microRNAs miR200c and miR34a demonstrate homology with the 3'UTR section of PDL-1 mRNA. Mir200c was recently shown to down regulate the expression of PD-L1 protein in a lung cancer model. Indeed, silencing of MUC-1 in AML cell lines MOLM-14 and THP-1 lead to a marked increase in miR200c and miR34a levels. Moreover, ectopic overexpression of these miRNAs resulted in near abrogation of PD-L1 expression. To examine if the increase in miR200c and miR34a was representative of a class effect on miRNAs, a miRNA NanoString array was performed in MUC1 silenced MOLM-14 and THP-1 cell lines. MUC1 silenced MOLM-14 cells showed an increase in 786/801 (98.1%) of miRNAs probed, of which 340 (42.4%) reached Bonferroni-corrected significance. Concordantly, MUC1 silenced THP-1 cells showed an increase in 698/801 (87.1%) of miRNAs probed, of which 154 (19.2%) reached Bonferroni-corrected significance, suggesting that MUC1 silencing resulted in a global increase in miRNA expression. miRNAs are processed from precursor (pre-) miRNAs to mature miRNAs by the proteins DICER and Argonaut-2. To elucidate the mechanism of MUC1-C regulation of miRNA expression, MUC1 silenced MOLM-14 and THP-1 cells were evaluated for their expression of pre-miR200c and pre-miR34a. The results demonstrated no change in the miRNA precursor levels, in contrast to the levels of mature miRNAs. This suggested that MUC1-C might regulate the processing of precursor miRNAs to their mature form. MUC1 silenced MOLM-14 and THP-1 demonstrated an increase in the mRNA and protein expression of DICER, but not Argonaut-2. To elucidate the mechanism of MUC1-C regulation of DICER expression, we examined transcription factors known to regulate DICER. cJUN is a key regulator of DICER expression that has been reported to be repressed by MUC1 signaling. Immunoblotting of MUC1 silenced MOLM14 and THP-1 demonstrated an increase c-JUN expression, and moreover, inhibition of cJUN activity in AML cells lines using a peptide inhibitor of c-JUN, abrogated DICER expression in a dose dependent manner. In conclusion, these findings strongly suggest MUC1-C as a key regulator of microRNA expression and demonstrate a critical mechanism by which MUC1-C signaling exploits noncoding RNAs to promote PD-L1 expression, resulting in an immunosuppressive characteristic of AML cells