Low CD49d expression in newly diagnosed chronic lymphocytic leukaemia may be associated with high-risk features and reduced treatment-free-intervals.

This study was carried out to assess the prognostic power of low CD49d expression (≥10%) in newly diagnosed CLL patients using a previously described cohort. Eighty-five patients were included. Median age at diagnosis; 70 years (43-88); CD49d was expressed in 33/85 (38.8%); 23/33 (69.7%) at ≥30% referred to as 'HiCD49d' and 10/33 (30.3%) between 10 and 30% with a bimodal pattern on scatterplot analysis referred to as 'LoCD49d'. Eleven patients (12.9%) presented as Binet stage B, of whom 8 (72.7%) were CD49d+ (HiCD49d 7/8; LoCD49d 1/8). Seven of 81 patients (8.6%) were NOTCH1 mutated and all were CD49d+ (p ≤ .01). IgVH analysis was performed on 29 (87.8%) of the CD49d+ cases, of whom 21 (72.4%) were unmutated and 8 (27.6%) were mutated. CD38+/CD49d+ accounted for 11/20 (55%) (CD38+/HiCD49D: 9/11; CD38+/LoCD49D: 2/11). At 42 months, treatment had been initiated in 18/85 (21%) patients, of these 10/33 (30.3%) were CD49d+ versus 8/52 (15.4%) of the CD49d- group. The median treatment free interval for the CD49d+ group was 11 months (HiCD49d; 14.5 months, LoCD49d; 11 months) compared to 21.5 months for the CD49d- group. These findings suggest that the predictive value of CD49d expression is retained at expression levels down to 10%.

COVID19 coagulopathy in Caucasian patients.

Although the pathophysiology underlying severe COVID19 remains poorly understood, accumulating data suggest that a lung-centric coagulopathy may play an important role. Elevated D-dimer levels which correlated inversely with overall survival were recently reported in Chinese cohort studies. Critically however, ethnicity has major effects on thrombotic risk, with a 3-4-fold lower risk in Chinese compared to Caucasians and a significantly higher risk in African-Americans. In this study, we investigated COVID19 coagulopathy in Caucasian patients. Our findings confirm that severe COVID19 infection is associated with a significant coagulopathy that correlates with disease severity. Importantly however, Caucasian COVID19 patients on low molecular weight heparin thromboprophylaxis rarely develop overt disseminated intravascular coagulation (DIC). In rare COVID19 cases where DIC does develop, it tends to be restricted to late-stage disease. Collectively, these data suggest that the diffuse bilateral pulmonary inflammation observed in COVID19 is associated with a novel pulmonary-specific vasculopathy termed pulmonary intravascular coagulopathy (PIC) as distinct to DIC. Given that thrombotic risk is significantly impacted by race, coupled with the accumulating evidence that coagulopathy is important in COVID19 pathogenesis, our findings raise the intriguing possibility that pulmonary vasculopathy may contribute to the unexplained differences that are beginning to emerge highlighting racial susceptibility to COVID19 mortality.

Splenectomy-related red cell lysis resistance causing analytical difficulty in a patient with a hematological malignancy.

A patient with a history of chronic lymphocytic leukaemia and a previous splenectomy underwent full blood count analysis in a general hospital. Her medical care had previously taken place in a different institution. A CELL- DYN Sapphire analyser measured her lymphocyte count at ten-fold higher than her known baseline. The sample was sent to her previous hospital, where the laboratory utilises an ADVIA-2120i analyser. The results of this analysis were in keeping with her baseline. The spurious result appears to be related to red cell lysis resistance following splenectomy; however, this resistance appeared to be specific to the analytical method used.

Does vaccine dose predict response to the monovalent pandemic H1N1 influenza a vaccine in children with acute lymphoblastic leukemia? A single-centre study.

BACKGROUND: Vaccination against influenza is an important strategy in preventing severe infection among children with acute lymphoblastic leukemia (ALL). Successful vaccination depends on both vaccine and host-related factors. We conducted a study on factors predicting the immunogenicity of the monovalent pandemic H1N1 (pH1N1) influenza A vaccine in children with ALL. METHODS: Children with ALL in our hospital were recruited and received two doses of the inactivated split-virion AS03-adjuvanted vaccine. The serological response was measured before each vaccine dose (Day 0 and 28) and 3 months after the second dose. Antibody titres were measured using a hemagglutination-inhibition assay. Seroconversion was defined as a ≥fourfold increase in antibody titre and a post-vaccination titre ≥1:40. RESULTS: Pre and post-vaccination titres were available from 45 children with ALL after one dose of the vaccine and 39 children after two doses. The seroconversion rate was 11.1% after one dose and 25.6% after the second dose. Univariate analysis demonstrated a significantly higher (P = 0.01) seroconversion rate among children who received the adult dose (0.5 ml) of the vaccine and a trend towards increased seroconversion (P = 0.07) by multivariate analysis. Factors including age, gender, lymphocyte count, treatment phase and regimen did not significantly affect the seroconversion rate. Children who received the adult dose demonstrated a significantly greater magnitude of serological response after both one dose (P = 0.04) and two doses (P = 0.001). CONCLUSIONS: These data suggest that the immunogenicity of the pH1N1 vaccine among children with ALL is improved by repeated and adult doses of the vaccine.

Severe COVID-19 is characterised by inflammation and immature myeloid cells early in disease progression.

SARS-CoV-2 infection causes a wide spectrum of disease severity. Identifying the immunological characteristics of severe disease and the risk factors for their development are important in the management of COVID-19. This study aimed to identify and rank clinical and immunological features associated with progression to severe COVID-19 in order to investigate an immunological signature of severe disease. One hundred and eight patients with positive SARS-CoV-2 PCR were recruited. Routine clinical and laboratory markers were measured, as well as myeloid and lymphoid whole-blood immunophenotyping and measurement of the pro-inflammatory cytokines IL-6 and soluble CD25. All analysis was carried out in a routine hospital diagnostic laboratory. Univariate analysis demonstrated that severe disease was most strongly associated with elevated CRP and IL-6, loss of DLA-DR expression on monocytes and CD10 expression on neutrophils. Unbiased machine learning demonstrated that these four features were strongly associated with severe disease, with an average prediction score for severe disease of 0.925. These results demonstrate that these four markers could be used to identify patients developing severe COVID-19 and allow timely delivery of therapeutics.

Acute Lymphoblastic Leukemia Arising in CALR Mutated Essential Thrombocythemia.

The development of acute lymphoblastic leukemia in an existing myeloproliferative neoplasm is rare with historical cases unable to differentiate between concomitant malignancies or leukemic transformation. Molecular studies of coexisting JAK2 V617F-positive myeloproliferative neoplasms and mature B cell malignancies indicate distinct disease entities arising in myeloid and lymphoid committed hematopoietic progenitor cells, respectively. Mutations of CALR in essential thrombocythemia appear to be associated with a distinct phenotype and a lower risk of thrombosis yet their impact on disease progression is less well defined. The as yet undescribed scenario of pro-B cell acute lymphoblastic leukemia arising in CALR mutated essential thrombocythemia is presented. Intensive treatment for the leukemia allowed for expansion of the original CALR mutated clone. Whether CALR mutations in myeloproliferative neoplasms predispose to the acquisition of additional malignancies, particularly lymphoproliferative disorders, is not yet known.

Valproate activates phosphodiesterase-mediated cAMP degradation: relevance to C6 glioma G1 phase progression.

Forskolin, a diterpene activator of adenylate cyclase, stimulates the production of cyclic adenosine monophosphate (cAMP) in a wide variety of cell types. In C6 glioma, used in this study, the anticonvulsant agent valproic acid (VPA) inhibited forskolin-stimulated cAMP accumulation in intact cells in a concentration-dependent manner. Kinetic studies indicated this valproate effect not to be mediated by direct inhibition of adenylate cyclase activity. The valproate-induced inhibition of cAMP accumulation was partially reversed by the phosphodiesterase (PDE) inhibitor isobutylmethyl xanthine (IBMX). Degradation of cAMP over time was more rapid in valproate-treated cells than in controls, and this effect was also reversed by IBMX. In synchronised C6 glioma, phosphodiesterase type IV (PDE4A1) expression was selectively upregulated during the G1 phase, in tandem with temporal biphasic peaks of cAMP. However, the expression of PDE4 isoforms was not affected by a 48-h exposure to valproate. These findings suggest inhibition of forskolin-stimulated cAMP levels in C6 glioma by valproate to be mediated by increased activation of PDE in the G1 phase. Since the degree of cell cycle arrest induced by valproate is intimately associated with its teratogenic potency, it appears that PDE-mediated inhibition of cAMP may contribute to the molecular mechanisms of valproate-induced teratogenicity.

Antiproliferative action of valproate is associated with aberrant expression and nuclear translocation of cyclin D3 during the C6 glioma G1 phase.

Cell cycle progression is tightly regulated by cyclins, cyclin-dependent kinases (cdks) and related inhibitory phophatases. Here, we employed mitotic selection to synchronize the C6 glioma cell cycle at the start of the G1 phase and mapped the temporal regulation of selected cyclins, cdks and inhibitory proteins throughout the 12 h of G1 by immunoblot analysis. The D-type cyclins, D3 and D1, were differentially expressed during the C6 glioma G1 phase. Cyclin D1 was up-regulated in the mid-G1 phase (4-6 h) while cyclin D3 expression emerged only in late G1 (9-12 h). The influence of the anticonvulsant agent valproic acid (VPA) on expression of cyclins and related proteins was determined, since its teratogenic potency has been linked to cell cycle arrest in the mid-G1 phase. Exposure of C6 glioma to VPA induced a marked up-regulation of cyclin D3 and decreased expression of the proliferating cell nuclear antigen. In synchronized cell populations, increased expression of cyclin D3 by VPA was detected in the mid-G1 phase (3-5 h). Immunocytochemical localization demonstrated rapid intracellular translocation of cyclin D3 to the nucleus following VPA exposure, suggesting that VPA-induced cell cycle arrest may be mediated by precocious activation of cyclin D3 in the G1 phase.

Automated in vitro screening of teratogens.

We present a new in vitro assay for screening of potential teratogens, based on staining of cultured mouse fibroblastoid L929 cells for the determination of number of live and dead cells and of cell morphology, employing automatic video recording, followed by detection of the stained specimen and calculation of endpoint values by the use of a computerized microscope workstation. Ten different parameters were combined empirically into a single index describing general alterations in cell morphology, and, subsequently, measurements of alterations in morphology and proliferation were combined to produce a single empirical index aimed at predicting teratogenic potency. The assay was employed in two different laboratories on 10 coded compounds; 7 compounds that have demonstrated in vivo teratogenic potentials: valproic acid (VPA), pentyl-4-yn-VPA, retinoic acid (RA), 13-cis-RA, AM580, thalidomide, and alpha-EM12 and 3 compounds for which no teratogenic potential has been demonstrated: isobutyl-4-yn-VPA, phytanic acid, and beta-EM12. Within each of the three groups of compounds the nonteratogens generally caused smaller alterations in cell morphology than the teratogens, although the effects of thalidomide and related compounds generally were minor or insignificant. The data support the hypothesis that cell morphology and proliferation in combination with other endpoints may be employed for in vitro screenings of potential teratogens, although studies of additional compounds are needed in order to establish the general validity of the procedure.