Standardization of 8-color flow cytometry across different flow cytometer instruments: A feasibility study in clinical laboratories in Switzerland.

The EuroFlow Consortium developed a fully standardized flow cytometric approach from instrument settings, through antibody panel, reagents and sample preparation protocols, to data acquisition and analysis. The Swiss Cytometry Society (SCS) promoted a study to evaluate the feasibility of using such standardized measurements of 8-color data across two different flow cytometry platforms - Becton Dickinson (BD) FACSCanto II and Beckman Coulter (BC) Navios, aiming at increasing reproducibility and inter-laboratory comparability of immunophenotypic data in clinical laboratories in Switzerland. The study was performed in two phases, i.e. a learning phase (round 1) and an analytical phase (rounds 2 and 3) consisting of a total of three rounds. Overall, 10 laboratories using BD FACSCanto II (n=6) or BC Navios (n=4) flow cytometers participated. Each laboratory measured peripheral blood samples from healthy donors stained with a uniform antibody panel of reagents - EuroFlow Lymphoid Screening Tube (LST) - applying the EuroFlow standardized protocols for instrument setup and sample preparation (www.EuroFlow.org). All data files were analyzed centrally and median fluorescence intensity (MedFI) values for individual markers on defined lymphocyte subsets were recorded; variability from reference MedFI values was assessed using performance scores. Data troubleshooting and discussion of the results with the participants followed after each round at SCS meetings. The results of the learning phase demonstrated that standardized instrument setup and data acquisition are feasible in routine clinical laboratories without previous experience with EuroFlow. During the analytical phase, highly comparable data were obtained at the different laboratories using either BD FACSCanto II or BC Navios. The coefficient of variation of MedFI for 7 of 11 markers performed repeatedly below 30%. In the last study round, 89% of participants scored over 90% MedFI values within the acceptance criteria (P-score), in line with the results of the EuroFlow quality assessment rounds performed by the EuroFlow expert laboratories(Kalina et al., 2015). Central analysis of data allowed identification of deviations from the standardized procedures and technical issues (e.g. failure to perform correct instrument setup and improper compensation). In summary, here we show that inter-laboratory cross-platform standardization of 8-color flow cytometric measurements in clinical laboratories is feasible and allows for fully comparable MedFI results across BD FACSCanto II and BC Navios instruments. However, adherence to standardized protocols is crucial. Thus, training of the laboratory personnel in the EuroFlow standardized procedures is highly recommended to prevent errors in instrument setup and sample preparation.

inv(16) and NPM1mut AMLs engraft human cytokine knock-in mice.

Favorable-risk human acute myeloid leukemia (AML) engrafts poorly in currently used immunodeficient mice, possibly because of insufficient environmental support of these leukemic entities. To address this limitation, we here transplanted primary human AML with isolated nucleophosmin (NPM1) mutation and AML with inv(16) in mice in which human versions of genes encoding cytokines important for myelopoiesis (macrophage colony-stimulating factor [M-CSF], interleukin-3, granulocyte-macrophage colony-stimulating factor, and thrombopoietin) were knocked into their respective mouse loci. NPM1mut AML engrafted with higher efficacy in cytokine knock-in (KI) mice and showed a trend toward higher bone marrow engraftment levels in comparison with NSG mice. inv(16) AML engrafted with high efficacy and was serially transplantable in cytokine KI mice but, in contrast, exhibited virtually no engraftment in NSG mice. Selected use of cytokine KI mice revealed that human M-CSF was required for inv(16) AML engraftment. Subsequent transcriptome profiling in an independent AML patient study cohort demonstrated high expression of M-CSF receptor and enrichment of M-CSF inducible genes in inv(16) AML cases. This study thus provides a first xenotransplantation mouse model for and informs on the disease biology of inv(16) AML.

Ultrasound presentation of abdominal non-Hodgkin lymphomas in pediatric patients.

INTRODUCTION: Burkitt's lymphoma accounts for approximately 25% of lymphomas diagnosed in children of developmental age. The tumor is localized mainly in the intestine (usually in the ileocecal region), mesenteric lymph nodes and extraperitoneal space. The clinical symptoms are non-specific and include: abdominal pain, vomiting, gastrointestinal bleeding, and acute abdomen suggesting appendicitis or intestinal intussusception. On ultrasound examination, Burkitt's lymphoma may manifest itself in various ways, depending on the origin of the lesion. AIM: The aim of this paper was to review the ultrasound manifestation of abdominal Burkitt's lymphoma in children. MATERIAL AND METHODS: The analysis included 15 pediatric patients with Burkitt's non-Hodgkin lymphoma in the abdominal cavity. The mean age of the patients was 9.5. Abdominal and gastrointestinal ultrasound examinations were conducted using a Siemens scanner with a convex transducer of 3.5-5 MHz and linear array transducer of L4 - 7.5 MHz. RESULTS: Ultrasound examinations conducted in the group of 15 patients revealed pathological masses localized in the gastric wall in 3 patients (20%), in the ileocecal region in 10 patients (67%) and a disseminated process in 2 patients (13%). In 12 patients with a diagnosed Burkitt's non-Hodgkin lymphoma in an extragastric localization, differences in the morphology of the lesions were observed. CONCLUSIONS: The clinical and ultrasound picture of abdominal Burkitt's lymphoma in children is variable. A careful ultrasound assessment of all abdominal organs conducted with the use of convex and linear probes increases the chances of establishing an adequate diagnosis.

[Malignant neoplasms localised in the parameningeal region in children treated at two Polish oncological centres-diagnostic and therapeutic dilemmas]. / Nowotwory zlosliwe o lokalizacji okolooponowej u dzieci w materiale dwóch osrodków onkologicznych--trudnosci diagnostyczne i terapeutyczne.

UNLABELLED: Malignant neoplasms localized in the parameningeal region include mainly soft tissue sarcomas (MTM), non-Hodgkin s lymphomas (NHL-B) and, less frequently, nasopharyngeal carcinomas. The aim of the study was to analyze diagnostic and therapeutic problems in children with parameniingeal neoplasms treated in Departments of Paediatric Oncology in Gdansk and Lublin between 1992 and 2004. MATERIAL AND METHODS: The study includes 32 patients (M/F: 23/9), aged 2 to 17 years, mean 6,3 years. In 17 children MTM was diagnosed: in nine NHL-B-cell and in six--nasopharyngeal carcinoma (lymphoepithelioma). The diagnosis of NHL-B and undifferentiated MTM were made in two children treated previously for NHL-nonB and retinoblastoma. Two cases of NHL appeared in a girl with ataxia-teleangiectasia syndrome. RESULTS: Initial symptoms lasted from 2 weeks to 24 months, mean 4,5 months for the whole group. In NHL patients mean period ofsymptoms was 4,5 weeks, in MTM- 5,5 months and in lynmphoepithelioma--7 months. Symptoms associated with the tumours localisation (snoring, breathing through the mouth, epistaxis, chronic purulent rhinitis, dysphagia and earache) predominated and were treated initially as upper respiratory tract infections. Cervical lymph nodes enlargement was observed in 30% children with MTM and 83% with lymphoepithelioma. Most of patients presented with highly advanced stages of neoplasms. MTM and NHL-B treatment was conducted according to the protocol approved by the Polish Paediatric Solid Tumours and Leukaemia/Lymphoma Studies Group. In patients with lymphoepithelioma different treatment schemes were administered, including chemo- and radiotherapy. Good response to therapy was found in 13/32 patients (41%). The group included 24% children with MTM (all with embryonic subtype), 56% with NHL-B and 67% with lymphoepithelioma. All these patients attained complete remission after standard line I therapy. But 13 children with MTM, four with NHL-B and two with lymphoepithelioma required more aggressive line II treatment because ofpoor response to therapy (NR) or relapse. Finally, 20 of 32 followed-up patients (62,5%) are in durable complete remission from 10 months to 11 years 4 months (mean 4 years) after therapy discontinuation. This group consists of all nine patients with NHL-B, 67% children with lymphoepithelioma and 41% with MTM. In six children (30%) persistent complications of oncological treatment occurred, including: hypoacusia, postradiation defect of the eye ball, postsurgical facial nerve palsy and cranio-nasal fistula complicated with pneumocephaly. A patient with MTM of maxillary sinus developed a second neoplasm 2 years after first therapy. This was glioblastoma multiforme located in the left parietal lobe (outside the radiation field). At present, the boy is in complete remission 2,5 years after treatment for the second tumour Among 32 children with parameningeal neoplasms 11 patients died (nine with MTM and two with lymphoepithelioma), all in the phase of disease progression (five NR and six after relapse). In two of them (with MTM) the direct cause of death was myelosupressive, gastrotoxic and infectious complications of antitumour therapy. One child still undergoes treatment for MTM relapse.

[Non-Hodgkin lymphomas of the nasopharynx in children--diagnostic and therapeutic difficulties]. / Chloniaki nieziarnicze nosogardla u dzieci--trudnosci diagnostyczne i terapeutyczne.

UNLABELLED: About 7% of all childhood cancers comprise non-Hodgkin's lymphoma (NHL). NHL are heterogenous group of neoplasms deriving from lymphatic system (cell B and T). B-cell NHL characterize by high malignancy, but coincidentally good reaction for treatment. In about 20% primary tumour is localised within head and neck, and nasopharynx lymphomas comprise 10%. This location maintains the biggest diagnostic and therapeutic difficulties because of tumours of this site proliferate in the region of frequent infections postponing a proper diagnosis and the local control after complete treatment is difficult. AIM OF THE STUDY: The authors analyse clinical symptoms before diagnosis, the incidence of nasopharynx lymphomas, histopathological type of neoplasm, clinical stage, results of treatment. MATERIAL AND METHODS: The study includes 97 patients who were treated because of lymphomas between 1993-2002. The character of clinical symptoms and their duration, histopathological type of lymphomas, results of treatment were analysed. RESULTS: The primary nasopharynx location was assessed in 9 patients (9.3%). Sex: 7 boys, 2 girls, age: 2-17 years. The duration which elapsed from initial clinical symptoms to diagnosis was 2-10 weeks. The histopathological assessment in 6 children was Burkitt lymphoma and in 3 children--Burkitt-like lymphoma. Metastases: CNS--1 patient, bone marrow--1 patient, abdomen--1 patient. Treatment was performed according to LMB-89 protocol. RESULTS: First complete remission--7 patients; second complete remission--2 patients. CONCLUSIONS: Lymphomas of nasopharynx cause diagnostic problems because of their early stage pseudo-inflammatory manifestation. Special attention should be paid to perform imaging studies (MRI/CT), which are useful in the reaching the proper diagnosis. The radiologic evaluation of primary lesion is still difficult. In the doubtful cases, the surgery and histopathological examination are necessary.

[Malignant pelvic neoplasms in children treated in two Polish oncology centres]. / Nowotwory zlosliwe miednicy mniejszej u dzieci w materiale dwóch osrodków onkologicznych w Polsce.

BACKGROUND: Extensive diagnostic and therapeutic dilemmas appear in children With primary malignant neoplasms located in the minor pelvis. THE AIM OF THE STUDY: To evaluate the clinical symptoms, disease course and the results of treatment in patients with malignant pelvic neoplasms. MATERIAL AND METHODS: The study included 31 children (13 boys and 18 girls; aged 2 months to 16 years; mean age -- 8 years) treated in the Departments of Paediatric Oncology and Haematology in Gdansk and Lublin during the period of 1992-2003. The group comprised 17 patients with soft tissue sarcomas (MTM) (55%), 12 with germinal tumours (TGM) (39%) and tow. with neuroblastoma (NBL) (6%). The great majority of children (90%) presented with highly advanced disease (stages III + IV -- in 28 out of 31 patients). RESULTS: with data analysis we were able to distinguish two categories of patients with different prognosis: with MTM and TGM. Most of he MTM tumours (11/17 - 65%) were localized in the urinary tract, the remaining six developed within pelvic muscles. Ten out of twelve TGM tumours (83%) were located in the ovaries. Radical tumour resection, especially primary resection, was shown to play the key role in both groups. Among TGM patients it was performed in 75% while in MTM patients -- in only 12%. All of these patients entered clinical remission and remain disease free. After adjuvant chemo- and/or radiotherapy secondary tumour resection was done in 17% of TGM and 41% of MTM patients. CONCLUSION: in patients, who were not able to undergo radical tumour resection (mainly MTM patients), the disease progressed and led to death.

Incidence of childhood cancers in Poland in 1995-1999.

BACKGROUND: The annual rate for childhood cancers in developed countries amounts to 105-130 new cases per 1 million children. The Polish population aged 0-17 years is estimated at approximately 10 million children and adolescents, thus ca. 1100-1300 new cases can be expected every year. In 1995, we started a national childhood cancer registry. MATERIAL/METHODS: Information on the new diagnoses of childhood cancers was collected in 11 regional centers and submitted to the national center in Lublin. All data were verified carefully and standardized incidence rates were calculated. RESULTS: In 1995, we registered 1028 newly diagnosed malignant neoplasms, in 1996 and 1997 - 1036 cases, in 1998 - 1007, and in 1999 - 1158 new cases. The estimated incidence rates were: 102.4; 109.5; 111.9; 111.6 and 118.3 per 1 million children, respectively. The most frequent childhood cancers include leukemia, which accounts for 28% of cancer cases, lymphoma (14.3%) and C. N. S. tumors (16.3%). CONCLUSIONS: Neoplasms of the hematopoietic system (leukemias and lymphomas) account for about 42% of all childhood cancers. Malignant lymphomas, bone tumors and germinal tumors are more frequently diagnosed in Poland, but the incidence of central nervous system tumors is lower than in other countries.