Signal intensity measured in a mass cytometry (CyTOF) channel can often be affected by the neighboring channels due to technological limitations. Such signal artifacts are known as spillover effects ...and can substantially limit the accuracy of cell population clustering. Current approaches reduce these effects by using additional beads for normalization purposes known as single‐stained controls. While effective in compensating for spillover effects, incorporating single‐stained controls can be costly and require customized panel design. This is especially evident when executing large‐scale immune profiling studies. We present a novel statistical method, named CytoSpill that independently quantifies and compensates the spillover effects in CyTOF data without requiring the use of single‐stained controls. Our method utilizes knowledge‐guided modeling and statistical techniques, such as finite mixture modeling and sequential quadratic programming, to achieve optimal error correction. We evaluated our method using five publicly available CyTOF datasets obtained from human peripheral blood mononuclear cells (PBMCs), C57BL/6J mouse bone marrow, healthy human bone marrow, chronic lymphocytic leukemia patient, and healthy human cord blood samples. In the PBMCs with known ground truth, our method achieved comparable results to experiments that incorporated single‐stained controls. In datasets without ground‐truth, our method not only reduced spillover on likely affected markers, but also led to the discovery of potentially novel subpopulations expressing functionally meaningful, cluster‐specific markers. CytoSpill (developed in R) will greatly enhance the execution of large‐scale cellular profiling of tumor immune microenvironment, development of novel immunotherapy, and the discovery of immune‐specific biomarkers. The implementation of our method can be found at https://github.com/KChen-lab/CytoSpill.git.
Department of Haematology, Imperial College London, Hammersmith Hospital, London, UK
Correspondence: Jamshid Sorouri-Khorashad, Department of Haematology, Hammersmith Hospital, Imperial College ...London W12 0NN, London, United Kingdom. E-mail: jamshid.sorouri-khorashad06{at}imperial.ac.uk
ABSTRACT
Imatinib is currently the first line therapy for newly diagnosed patients with chronic myeloid leukemia. However, 20–25% of patients do not achieve durable complete cytogenetic responses. The mechanism underlying this primary resistance is unknown, but variations in BCR-ABL1 kinase activity may play a role and can be investigated by measuring the autophosphorylation levels of BCR-ABL1 or of a surrogate target such as Crkl. In this study we used flow cytometry to investigate the in vitro inhibition of Crkl phosphorylation by imatinib in CD34 + cells in diagnostic samples from two groups of patients distinguished by their cytogenetic response. No difference in inhibition of Crkl phosphorylation was observed in the two groups. The observation that increasing the dose of imatinib in vivo did not increase the level of cytogenetic response in some non-responders suggests that in at least a proportion of patients imatinib resistance may be due to activation of BCR-ABL1-independent pathway.
Key words: CD34, CML, p-Crkl, imatinib.
Invariant natural killer T (iNKT) cells are powerful immunomodulatory cells that in mice regulate a variety of immune responses, including acute GVHD (aGVHD). However, their clinical relevance and in ...particular their role in clinical aGVHD are not known. We studied whether peripheral blood stem cell (PBSC) graft iNKT-cell dose affects on the occurrence of clinically significant grade II-IV aGVHD in patients (n = 57) undergoing sibling, HLA-identical allogeneic HSCT. In multivariate analysis, CD4− iNKT-cell dose was the only graft parameter to predict clinically significant aGVHD. The cumulative incidence of grade II-IV aGVHD in patients receiving CD4− iNKT-cell doses above and below the median were 24.2% and 71.4%, respectively (P = .0008); low CD4− iNKT-cell dose was associated with a relative risk of grade II-IV aGVHD of 4.27 (P = .0023; 95% CI, 1.68-10.85). Consistent with a role of iNKT cells in regulating aGVHD, in mixed lymphocyte reaction assays, CD4− iNKT cells effectively suppressed T-cell proliferation and IFN-γ secretion in a contact-dependent manner. In conclusion, higher doses of CD4− iNKT cells in PBSC grafts are associated with protection from aGVHD. This effect could be harnessed for prevention of aGVHD.
Most patients with high CMV-CMI and low-level CMV reactivation did not progress to clinically significant CMV infection. Low CMV-CMI and steroids use were the only predictors for CMV progression from ...low-level reactivation.
Abstract
Background
Cytomegalovirus (CMV) infections in hematopoietic cell transplant (HCT) recipients cause substantial morbidity and mortality. CMV cell-mediated immunity (CMV-CMI) can be determined by levels of interferon gamma (IFN-γ) production using an enzyme-linked immunospot (ELISPOT) CMV assay (T-SPOT.CMV assay). In this study, we evaluated the ability of this assay to predict the outcome of low-level CMV reactivation in HCT recipients.
Methods
We followed 55 HCT recipients with low-level CMV reactivation up to 8 weeks from enrollment. Progression to clinically significant CMV infection (CS-CMVi) was defined as a CMV load >1000 IU/mL or > 500 IU/mL in patients receiving matched related/autologous or matched unrelated transplants, respectively, and initiation of antiviral treatment.
Results
Progression to CS-CMVi occurred in 31 (56%) of the HCT recipients. Spot counts of CMV-specific pp65 and IE1 antigens were significantly lower in patients who had CS-CMVi than in patients who did not. On multivariate analysis, the ELISPOT CMV responses and steroids use were the only predictors of progression to CS-CMVi.
Conclusions
A strong association between low CMV-CMI and progression to CS-CMVi was observed in HCT recipients. The implementation of serial monitoring of CMV-CMI may identify patients at risk of progression to CS-CMVi that require antiviral therapy.
Abstract 3290
Poster Board III-1▪▪This icon denotes an abstract that is clinically relevant.
There is a great variability in the degree of molecular responses achieved by chronic myeloid leukemia ...(CML) patients treated with imatinib. These different levels of molecular response could reflect different degrees of adherence to therapy. We measured the adherence to imatinib therapy in 87 consecutive CML chronic phase patients who had received imatinib 400 mg day as first line therapy for a median of 59.7 months before enrolment (range 25–104) and therefore all them were in complete cytogenetic response. Adherence levels were monitored during a 3-month period using microelectronic monitoring devices (MEMS) and were related to levels of molecular response. MEMS consist of an electronic device fitted in the cap of a normal looking medication bottle that automatically records each time the bottle is opened. MEMS are considered as the ‘gold standard' for measuring adherence. We also measured the imatinib plasma level, the presence of TKD mutations and the following prognostic factors measured at diagnosis: hOCT1 transcripts level, polymorphism 1236C>T in ABCB1, Sokal risk group, hemoglobin, leukocytes , BCR-ABL1 transcript type and BCR1-ABL1 ratio and demographic data. The study protocol was approved by the Research Ethics Committee and patients gave written informed consent to participate.
The median adherence rate was 97.6% (range 22.6–103.8%). In 23 (26.4%) patients adherence was ≤90% (median 76%) and in 12 (13.8%) ≤80% (median 63%). We found a strong association between adherence rate (≤90% or >90%) and the 6-year probability of major molecular response (MMR) (28.4% vs 94.5%, p<0.0001) and complete molecular response (CMR) (0% vs 43.8%, p=0.002) (Fig 1). Multivariate analysis identified adherence (RR=11.7, p=0.001) and expression of the molecular transporter hOCT1, (RR=1.79, p=0.038) as the only independent predictors for MMR. Adherence was the sole independent predictor for CMR. No molecular responses were observed when the adherence was ≤20% (p=0.0001). In patients whose imatinib dose had been increased (n=32) the adherence was poor (median 86.4%). Adherence was the only independent predictor for failure to achieve a 3-log transcript reduction (RR=17.66, p=0.006) in this subgroup of patients. Patients with CML vary greatly in their response, as demonstrated originally by Sokal et al. in 1984, and the same variation is seen in patients treated with imatinib in the modern era. The basis for this variation is unknown but it has been attributed to the intrinsic biological heterogeneity of the leukemia. In contrast we show here that adherence to therapy is the major factor determining the degree of response that a CML patient treated with imatinib will achieve.
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Mahon:Novartis: Consultancy, Research Funding. Apperley:Novartis: Consultancy, Honoraria. Rezvani:Novartis: Consultancy, Honoraria, Research Funding. Marin:Novartis: Consultancy, Research Funding.
High grade epithelial ovarian carcinoma is an aggressive tumor. Treatment includes platinum therapy, however it recurs in most patients due to therapy resistance. In this project, we study the ...immunohistochemical (IHC) expression of five potential biomarkers/prognostic markers in high grade epithelial ovarian carcinoma: EGFR, HLA-G, CD70, c-MET, and NY-ESO1. A cohort of 274 patients is used. We compare the IHC expression with age, stage, ascites status, family history of cancer, disease free survival (DFS) and overall survival (OS). EGFR expression is significantly correlated with family history and worse OS. HLA-G is associated with worse OS. To confirm the results of EGFR and HLA-G, a second separated cohort of 248 patients is used. Positive EGFR expression again shows worse OS, while HLA-G expression has worse prognostic trend. CD70 has a worse OS trend. C-MET and NY-ESO1 do not have any clinical correlations. EGFR can potentially serve as target in future clinical immune therapy trials.
Abstract
Background
Recurrent pediatric medulloblastoma and ependymoma have a grim prognosis. We report a first-in-human, phase I study of intraventricular infusions of ex vivo expanded autologous ...natural killer (NK) cells in these tumors, with correlative studies.
Methods
Twelve patients were enrolled, 9 received protocol therapy up to 3 infusions weekly, in escalating doses from 3 × 106 to 3 × 108 NK cells/m2/infusion, for up to 3 cycles. Cerebrospinal fluid (CSF) was obtained for cellular profile, persistence, and phenotypic analysis of NK cells. Radiomic characterization on pretreatment MRI scans was performed in 7 patients, to develop a non-invasive imaging-based signature.
Results
Primary objectives of NK cell harvest, expansion, release, and safety of 112 intraventricular infusions of NK cells were achieved in all 9 patients. There were no dose-limiting toxicities. All patients showed progressive disease (PD), except 1 patient showed stable disease for one month at end of study follow-up. Another patient had transient radiographic response of the intraventricular tumor after 5 infusions of NK cell before progressing to PD. At higher dose levels, NK cells increased in the CSF during treatment with repetitive infusions (mean 11.6-fold). Frequent infusions of NK cells resulted in CSF pleocytosis. Radiomic signatures were profiled in 7 patients, evaluating ability to predict upfront radiographic changes, although they did not attain statistical significance.
Conclusions
This study demonstrated feasibility of production and safety of intraventricular infusions of autologous NK cells. These findings support further investigation of locoregional NK cell infusions in children with brain malignancies.
The cancer-risk-associated rs6983267 single nucleotide polymorphism (SNP) and the accompanying long noncoding RNA
in the highly amplified 8q24.21 region have been implicated in cancer predisposition, ...although causality has not been established. Here, using allele-specific
transgenic mice, we demonstrate that
overexpression leads to spontaneous myeloid malignancies. We further identified that
is overexpressed in bone marrow and peripheral blood of myelodysplastic/myeloproliferative neoplasms (MDS/MPN) patients.
induces global deregulation of gene expression by down-regulating EZH2 in vitro and in vivo in an allele-specific manner. We also identified a novel non-APOBEC, non-ADAR, RNA editing at the SNP locus in MDS/MPN patients and
-transgenic mice. The RNA transcribed from the SNP locus in malignant hematopoietic cells have different allelic composition from the corresponding genomic DNA, a phenomenon rarely observed in normal cells. Our findings provide fundamental insights into the functional role of rs6983267 SNP and
in myeloid malignancies.
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