scholarly journals Flow Cytometry Immunophenotyping for Diagnostic Orientation and Classification of Pediatric Cancer Based on the EuroFlow Solid Tumor Orientation Tube (STOT)

Cancers ◽  
2021 ◽  
Vol 13 (19) ◽  
pp. 4945
Author(s):  
Cristiane de Sá de Sá Ferreira-Facio ◽  
Vitor Botafogo ◽  
Patrícia Mello Ferrão ◽  
Maria Clara Canellas ◽  
Cristiane B. Milito ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Solid Tumors ◽  
Solid Tumor ◽  
Pediatric Cancer ◽  
Diagnostic Procedures ◽  
Hematologic Malignancies ◽  
Multiparameter Flow Cytometry ◽  
Diagnostic Screening ◽  
Pediatric Solid Tumors

Early diagnosis of pediatric cancer is key for adequate patient management and improved outcome. Although multiparameter flow cytometry (MFC) has proven of great utility in the diagnosis and classification of hematologic malignancies, its application to non-hematopoietic pediatric tumors remains limited. Here we designed and prospectively validated a new single eight-color antibody combination—solid tumor orientation tube, STOT—for diagnostic screening of pediatric cancer by MFC. A total of 476 samples (139 tumor mass, 138 bone marrow, 86 lymph node, 58 peripheral blood, and 55 other body fluid samples) from 296 patients with diagnostic suspicion of pediatric cancer were analyzed by MFC vs. conventional diagnostic procedures. STOT was designed after several design–test–evaluate–redesign cycles based on a large panel of monoclonal antibody combinations tested on 301 samples. In its final version, STOT consists of a single 8-color/12-marker antibody combination (CD99-CD8/numyogenin/CD4-EpCAM/CD56/GD2/smCD3-CD19/cyCD3-CD271/CD45). Prospective validation of STOT in 149 samples showed concordant results with the patient WHO/ICCC-3 diagnosis in 138/149 cases (92.6%). These included: 63/63 (100%) reactive/disease-free samples, 43/44 (98%) malignant and 4/4 (100%) benign non-hematopoietic tumors together with 28/38 (74%) leukemia/lymphoma cases; the only exception was Hodgkin lymphoma that required additional markers to be stained. In addition, STOT allowed accurate discrimination among the four most common subtypes of malignant CD45− CD56++ non-hematopoietic solid tumors: 13/13 (GD2++ numyogenin− CD271−/+ nuMyoD1− CD99− EpCAM−) neuroblastoma samples, 5/5 (GD2− numyogenin++ CD271++ nuMyoD1++ CD99−/+ EpCAM−) rhabdomyosarcomas, 2/2 (GD2−/+ numyogenin− CD271+ nuMyoD1− CD99+ EpCAM−) Ewing sarcoma family of tumors, and 7/7 (GD2− numyogenin− CD271+ nuMyoD1− CD99− EpCAM+) Wilms tumors. In summary, here we designed and validated a new standardized antibody combination and MFC assay for diagnostic screening of pediatric solid tumors that might contribute to fast and accurate diagnostic orientation and classification of pediatric cancer in routine clinical practice.

scholarly journals Contribution of Multiparameter Flow Cytometry Immunophenotyping to the Diagnostic Screening and Classification of Pediatric Cancer

PLoS ONE ◽  
2013 ◽  
Vol 8 (3) ◽  
pp. e55534 ◽  
Author(s):  
Cristiane S. Ferreira-Facio ◽  
Cristiane Milito ◽  
Vitor Botafogo ◽  
Marcela Fontana ◽  
Leandro S. Thiago ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Pediatric Cancer ◽  
Multiparameter Flow Cytometry ◽  
Diagnostic Screening

scholarly journals Hematologist‐Level Classification of Mature B‐Cell Neoplasm Using Deep Learning on Multiparameter Flow Cytometry Data

2020 ◽  
Vol 97 (10) ◽  
pp. 1073-1080
Author(s):  
Max Zhao ◽  
Nanditha Mallesh ◽  
Alexander Höllein ◽  
Richard Schabath ◽  
Claudia Haferlach ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Deep Learning ◽  
B Cell ◽  
Multiparameter Flow Cytometry ◽  
Flow Cytometry Data ◽  
Cell Neoplasm

Immunotherapy in Gynecologic Cancers: What We Know Now and Where We Are Headed

2019 ◽  
pp. e126-e140 ◽  
Author(s):  
Kimberly Levinson ◽  
Oliver Dorigo ◽  
Krista Rubin ◽  
Kathleen Moore
Keyword(s):  
Solid Tumors ◽  
Solid Tumor ◽  
Immune Checkpoint ◽  
Immune Checkpoint Inhibitors ◽  
Checkpoint Inhibitors ◽  
Gynecologic Cancer ◽  
Hematologic Malignancies ◽  
Next Generation ◽  
Gynecologic Cancers ◽  
Clinical Benefits

Immunotherapy, mainly in the form of immune checkpoint inhibitors (ICIs), has been transformative in both solid tumor and hematologic malignancies. Patients with previously terminal illnesses have experienced profound responses of great durability with these agents, fueling excitement among patients and providers regarding their use. Unfortunately, the gains seen in some solid tumors have not been replicated in a large percentage of patients with gynecologic cancer. This review focuses on the clinical benefits seen to date, toxicities and management when using ICIs, ways to improve prediction of who should receive immunotherapy, and a discussion of next-generation immunotherapy with cellular therapeutics and how these might relate to gynecologic cancers.

A biomarker-guided approach to combining PARP inhibitors with radiotherapy in pediatric solid tumors.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. 10556-10556
Author(s):  
Anang Shelat ◽  
Christopher Tinkle ◽  
Elizabeth Stewart ◽  
Sara Michele Federico ◽  
Brandon Bianski ◽  
...  
Keyword(s):  
Solid Tumors ◽  
Tumor Cells ◽  
Dna Adducts ◽  
Solid Tumor ◽  
Parp Inhibitors ◽  
Parp Inhibition ◽  
Drug Induced ◽  
Potent Inducer ◽  
Pediatric Solid Tumors ◽  
Pediatric Solid Tumor

10556 Background: Ewing sarcoma (EWS) expresses high levels of Schlafen-11 (SLFN11). SLFN11 disrupts checkpoint maintenance and may serve as a biomarker to assess sensitivity to Poly (ADP-ribose) polymerase 1 and 2 inhibitors (PARPi). The goal of this study is to evaluate SLFN11 protein expression in a panel of pediatric solid tumors and correlate levels of protein with sensitivity to PARP inhibition combined with ionizing radiation (IR), a component of therapy for many pediatric solid tumors and a potent inducer of DNA damage. Methods: SLFN11 mRNA and protein levels were assessed by quantitative RT-PCR, and immunohistochemistry, Western blot, and immunofluorescence microscopy, respectively. PARPi included: talazoparib (TAL), niraparib (NIR), veliparib (VEL), and olaparib (OLA). Approximately 30 minutes after addition of systemic therapy, graded doses of radiation were delivered and viability across a panel of pediatric solid tumor cell lines was measured using the ATP-based Cell TiterGlo assay and confirmed with the colony formation assay. Results: We found that SLFN11 mRNA and protein is expressed at high levels in EWS, and SLFN11 is also variably present in a subset of other pediatric solid tumor lines, including desmoplastic small round cell tumor, osteosarcoma, and rhabdomyosarcoma. In all tumor cells with detectable SLFN11 expression, viability was reduced by greater than 90% when exposed to 2Gy IR and 1-10nM TAL, whereas cells with undetectable levels of SLFN11 were 5-10 times less sensitive. Intriguingly, variation in the potentiation between specific PARPi and IR correlated with the ability to form drug-induced PARP-DNA adducts, with the strong PARP trapper TAL showing ~10-fold higher potency compared to the moderate trapper NIR, and ~300-fold more potency relative to the weak trapper VEL. Consistent with our PARPi findings, the toposiomerase 1 inhibitor irinotecan, which also forms DNA adducts, potentiated with IR similarly to TAL at concentrations < 10nM in tumor cells expressing detectable levels of SLFN11. Conclusions: SLFN11 is present in select pediatric solid tumors and may induce a DNA repair defect that is best exploited by combining low-doses of TAL and irinotecan with IR.

Prospective agnostic germline testing in pediatric cancer patients.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. 1589-1589
Author(s):  
Elise Fiala ◽  
Jennifer Kennedy ◽  
Yelena Kemel ◽  
Audrey Mauguen ◽  
Diana Mandelker ◽  
...  
Keyword(s):  
Cancer Patients ◽  
Solid Tumors ◽  
Pediatric Cancer ◽  
Spinal Tumors ◽  
Cancer History ◽  
Pediatric Solid Tumors ◽  
Pediatric Cancer Patients ◽  
High Penetrance ◽  
Tumor Types ◽  
Dominant Genes

1589 Background: We report our large cohort of pediatric cancer patients undergoing prospective agnostic germline sequencing. Our dataset is a significant addition to the 1,573 children reported to date who have undergone agnostic germline sequencing in previous large sequencing studies, each with ascertainment bias. Methods: 676 patients with pediatric solid tumors underwent matched tumor-normal targeted DNA sequencing from July 2015 to February 2020. At least 76 genes associated with cancer predisposition were analyzed in the germline, and variants were classified per American College of Medical Genetics guidelines. Pathogenic and likely pathogenic (P/LP) variants were reported to patients/families, who were offered genetic counseling and cascade testing with screening recommendations and referral to a surveillance clinic as appropriate. Results: One or more P/LP variants were found in 17% (115/676) of individuals when including low, moderate and high penetrance mutations in recessive and dominant genes, or 12% (81/676) when including moderate and high penetrance mutations in dominant genes. P/LP variants were detected in 40% (21/53) of patients with retinoblastomas, 8% (13/161) with neuroblastomas/ganglioneuroblastomas, 13% (14/112) with brain/spinal tumors, 8% (20/245) with sarcomas, and 12% (13/105) with other solid tumors. The most frequent mutations were in RB1 (n = 28) and TP53 (n = 8) in patients with associated tumors. Of patients with moderate/high penetrance mutations, 30% (24/81) had unexpected tumor types, with potential therapeutic relevance in 58% (14/24) including BRCA1 n = 2, BRCA2 n = 3, RAD51D n = 1, ATM n = 1 MLH1 n = 1, MSH2 n = 1, MSH6 n = 1, PMS2 n = 3, and SUFU n = 1. Two patients received immunotherapy based on their germline finding. Conclusions: P/LP germline variants are frequently present in patients with pediatric cancer. We are contributing significantly to the cohort size of agnostic sequencing in pediatric cancers. Our experience is similar to other studies with a ~12% detection rate of moderate and high penetrance mutations. Moderate/high penetrance mutations were concordant with the patient’s cancer history in 70% of cases, higher than previously reported, likely due to an enrichment of retinoblastoma. While many mutations are identified in patients with associated tumor types, a large proportion of mutations are unexpected based on the patient’s history. Clinical actionability of these findings may include screening, risk reduction, family planning, and increasingly targeted therapies.

scholarly journals Hematologist-level classification of mature B-cell neoplasm using deep learning on multiparameter flow cytometry data

2020 ◽  
Author(s):  
Max Zhao ◽  
Nanditha Mallesh ◽  
Richard Schabath ◽  
Alexander Höllein ◽  
Claudia Haferlach ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cell ◽  
Lymphocytic Leukemia ◽  
Multiparameter Flow Cytometry ◽  
Self Organizing Map ◽  
Prolymphocytic Leukemia ◽  
Flow Cytometry Data ◽  
Mantle Cell ◽  
Cell Neoplasm

AbstractThe wealth of information captured by multiparameter flow cytometry (MFC) can be analyzed by recent methods of computer vision when represented as a single image file. We therefore transformed MFC raw data into a multicolor 2D image by a self-organizing map (SOM) and classified this representation using a convolutional neural network (CNN). By this means, we built an artificial intelligence that is not only able to distinguish diseased from healthy samples, but that can also differentiate seven subtypes of mature B-cell neoplasm (B-NHL). We trained our model with 18,274 cases including chronic lymphocytic leukemia (CLL) and its precursor monoclonal B-cell lymphocytosis (MBL), marginal zone lymphoma (MZL), mantle cell lymphoma (MCL), prolymphocytic leukemia (PL), follicular lymphoma (FL), hairy cell leukemia (HCL), lymphoplasmacytic lymphoma (LPL) and achieved a weighted F1 score of 0.94 on a separate test set of 2,348 cases. Furthermore, we estimated the trustworthiness of a classification and could classify 70% of all cases with a confidence of 0.95 and higher. Our performance analyses indicate that particularly for rare subtypes further improvement can be expected when even more samples are available for training.

scholarly journals THE INTRA-ASSAY PRECISION AND TIME STABILITY OF QUANTITATIVE βIII-TUBULIN EXPRESSION ASSESSMENT IN SOLID TUMOR TISSUE

2020 ◽  
Vol 19 (3) ◽  
pp. 52-56
Author(s):  
A. A. Basharina ◽  
T. A. Bogush ◽  
E. A. Rukavishnikova ◽  
E. A. Bogush ◽  
S. A. Kaliuzhny ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Solid Tumors ◽  
Solid Tumor ◽  
Tumor Tissue ◽  
Quantitative Estimation ◽  
Flow Cytometer ◽  
Time Stability ◽  
Analytical Validation ◽  
Assay Precision ◽  
Solid Tumor Tissue

Introduction. The introducing of tumor molecular profiling into clinical practice has revealed the need for development of new analytical methods for estimating marker expression in solid tumors, as routinely used method of immunohistochemistry has a number of significant drawbacks.Objective. Analytical validation of immunofluorescence staining and flow cytometry method developed by the authors for the examination of tumor protein markers in the solid tumors tissue.Materials and methods. Method validation was carried out by quantitative estimation of βIII-tubulin (TUBB3) expression in single-cell suspensions of non-small-cell lung cancer obtained from surgical tumor samples. Primary antibodies to TUBB3 (ab7751) and secondary DyLight 650-conjugated antibodies (ab98729) were used for immunofluorescent staining. The «Navios» flow cytometer (Beckman Coulter) was used to measure the fluorescence. The validation parameters were assessed by the coefficient of variation calculated as the ratio of standard deviation of TUBB3 level to its mean value.Results. Two parameters were analyzed: intra-assay precision and time stability of the results of the TUBB3 expression assessment. It was demonstrated that the mean coefficients of variation of the marker expression level in the tumor tissue did not exceed 20 % for both parameters. According to recommendations on the analytical validation of methods based on flow cytometry, it proves the validity of the method for these parameters.Conclusions. The intra-assay precision and time stability were demonstrated for the results of a quantitative estimation of TUBB3 expression in solid tumor tissue using immunofluorescence staining and flow cytometry method developed by the authors. The practical value of the time stability of immunofluorescence stain during 24 h storage of a stained cells suspension in the dark at 4 °C was highlighted. It shows the possibility of adjusting the time interval between completion of the analytical study part and flow cytometer measurement.

Clinical Trial of NPI-0052 (2nd generation proteasome inhibitor) in Patients Having Advanced Malignancies with Expanded RP2D Cohorts in Lymphoma and CLL

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4934-4934 ◽  
Author(s):  
Timothy Price ◽  
Peeter Padrik ◽  
Amanda Townsend ◽  
Paul Mainwaring ◽  
Lawrence Catley ◽  
...  
Keyword(s):  
Solid Tumors ◽  
Solid Tumor ◽  
Proteasome Inhibitor ◽  
Standard Of Care ◽  
Proteasome Inhibition ◽  
Hematologic Malignancies ◽  
Preclinical Research ◽  
Novel Structure ◽  
Advanced Malignancies ◽  
Mantle Cell

Abstract Background: NPI-0052 is a novel proteasome inhibitor that produces prolonged inhibition of the 20S proteasome. NPI-0052 has a novel structure leading to a unique proteasome inhibition, toxicology and effect profile. Preclinical research suggests an improved therapeutic ratio and activity in hematologic and solid tumor malignancy models. Secondary to these findings, clinical trials are being conducted in patients with myeloma, lymphomas, leukemias and solid tumors. Materials and Methods: In this study patients with solid tumor, lymphoma or leukemia diagnoses were treated with NPI-0052 administered weekly, for 3 weeks in 4-week cycles in a 3+3 design dose escalation. The dose of NPI-0052 was escalated in 50–100% increments dependent on observed adverse events (AE). In addition to regular safety monitoring, proteasome inhibition (PI) (baseline, D1 & D15) and pharmacokinetics PK (D1 & D15) were assayed in blood. Once a Recommended Phase 2 Dose (RP2D) is identified, RP2D cohorts of 10 patients in each lymphoma and CLL are enrolled. Results: 25 patients have been treated at doses ranging from 0.1 mg/m2 to 0.7 mg/m2. The AE profile has been very tolerable with fatigue, transient peri-infusion site discomfort and lymphopenia being commonly ascribed to NPI-0052. Whole blood pharmacokinetics were calculated for all patients on study. At the highest dose assessed PK parameters were (mean ± SD) AUCtot =215±129 ng/mL*min; Cmax =22.8 ±14 ng/mL; t1/2 =13.5 ± 9.2 mins; clearance= 7.8 ± 8.2 L/min and Vss = 132 ± 192 L. AUC and Cmax increased linearly with dose and the kinetics are apparently not dose dependant. PI has been assayed in blood, indicating a dose:response relationship with inhibition of chymotrypsin-like activity up to 100% observed and mean Day 1 inhibition of 78%. This level of proteasome inhibition is higher than that reported with standard doses of bortezomib, yet the profile of adverse drug reactions associated with bortezomib has not been observed. A total of 7 patients (33%) have had stable disease for at least 2 cycles (8 weeks; 2months), including one each with mantle cell lymphoma (4 cycles), Hodgkin’s lymphoma (4 cycles), follicular lymphoma (4 cycles) and sarcoma (5 cycles) and prostate adenocarcinoma, and two with melanoma (4 cycles). Conclusions: NPI-0052 produces dose-dependent pharmacologic effects through the predicted efficacious range while producing a toxicity profile that is tolerable and dissimilar to that of the standard of care proteasome inhibitor bortezomib. These data have supported additional studies being initiated in hematologic malignancies and solid tumors.

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