The reliability of bone marrow cytology as response criterion in metastatic neuroblastoma.

BACKGROUND: The quantitative assessment of neuroblastoma cell content in bone marrow aspirates for response evaluation has been introduced recently. Data on the concordance of interobserver reports are lacking so far. METHODS: Investigators of seven European countries representing national reference or large oncological centers convened in 2016. They agreed to quantitatively assess routine bone marrow smears of the participating institutions and to discuss the discrepant results in joint meetings. RESULTS: From 2017 through 2019, three cytology rounds with 24, 28, and 28 bone marrow samples were run evaluating the representativity of the smears (yes/[restricted]/no) and the presence of tumor cells (yes/no and %). The comparison of the reports using κ (Fleiss) and α (Krippendorff) statistics demonstrated no robust reliabilities. The agreement on the representativity was moderate to poor, on the presence of tumor cells moderate to good, and on the percentage of tumor cells slight to moderate. Though the value of cytology is unquestioned to detect even tiny metastatic cells in bone marrow, the investigators unanimously agreed that a reliable quantification of the tumor cell content in bone marrow smears is unrealistic. For the key issue of representativity, a new practical definition was developed. CONCLUSION: For any work with bone marrow aspirates, the representativity of the material is of paramount importance. A practical definition is proposed. A reliable quantitative cytological assessment of tumor cell content in bone marrow aspirates is not feasible in metastatic neuroblastoma. Therefore, its use as response criterion should be reconsidered.

Subclinical myopathy in a child with neutral lipid storage disease and mutations in the PNPLA2 gene.

We report a 14-year-old-boy with markedly elevated serum creatine kinase (CK) levels, in whom massive triglyceride storage was found in peripheral blood leukocytes and in muscle biopsy. Sequencing PNPLA2, the gene encoding the adipose triglyceride lipase (ATGL) and responsible for the neutral lipid storage disease with myopathy (NLSDM), we identified two heterozygous mutations, including a previously reported nonsense and a novel missense mutation in the patatin domain of the gene. Lipid storage myopathy can be clinically silent in childhood and presenting only with hyperCKemia.

Multiparametric flow cytometry highlights B7-H3 as a novel diagnostic/therapeutic target in GD2neg/low neuroblastoma variants.

BACKGROUND: High-risk neuroblastomas (HR-NBs) are rare, aggressive pediatric cancers characterized by resistance to therapy and relapse in more than 30% of cases, despite using an aggressive therapeutic protocol including targeting of GD2. The mechanisms responsible for therapy resistance are unclear and might include the presence of GD2neg/low NB variants and/or the expression of immune checkpoint ligands such as B7-H3. METHOD: Here, we describe a multiparametric flow cytometry (MFC) combining the acquisition of 106 nucleated singlets, Syto16pos CD45neg CD56pos cells, and the analysis of GD2 and B7-H3 surface expression. 41 bone marrow (BM) aspirates from 25 patients with NB, at the onset or relapse, are analyzed, comparing results with cytomorphological analysis (CA) and/or immunohistochemistry (IHC). Spike in experiments assesses the sensitivity of MFC. Kaplan-Meier analysis on 498 primary NBs selects novel prognostic markers possibly integrating the MFC panel. RESULTS: No false positive are detected, and MFC shows high sensitivity (0.0005%). Optimized MFC identifies CD45negCD56pos NB cells in 11 out of 12 (91.6%) of BM indicated as infiltrated by CA, 7 of which coexpress high levels of GD2 and B7-H3. MFC detects CD45negCD56posGD2neg/low NB variants expressing high surface levels of B7-H3 in two patients with HR-NB (stage M) diagnosed at 53 and 139 months of age. One of them has a non-MYCN amplified tumor with unusual THpos PHOX2Bneg phenotype, which relapsed 141 months post-diagnosis with BM infiltration and a humerus lesion. All GD2neg/low NB variants are detected in patients at relapse. Kaplan-Meier analysis highlights an interesting dichotomous prognostic value of MML5, ULBPs, PVR, B7-H6, and CD47, ligands involved in NB recognition by the immune system. CONCLUSIONS: Our study validates a sensitive MFC analysis providing information on GD2 and B7-H3 surface expression and allowing fast, specific and sensitive evaluation of BM tumor burden. With other routinely used diagnostic and prognostic tools, MFC can improve diagnosis, prognosis, orienting novel personalized treatments in patients with GD2low/neg NB, who might benefit from innovative therapies combining B7-H3 targeting.

Clinical and morphological practices in the diagnosis of transplant-associated microangiopathy: a study on behalf of Transplant Complications Working Party of the EBMT.

Transplant-associated thrombotic microangiopathy (TA-TMA) is a life-threatening complication of allogeneic hematopoietic stem cell transplantation (HSCT). This study evaluated clinical and morphological practices of TA-TMA diagnosis in EBMT centers. Two questionnaires, one for transplant physician and one for morphologist, and also a set of electronic blood slides from 10 patients with TA-TMA and 10 control patients with various erythrocyte abnormalities, were implemented for evaluation. Seventeen EBMT centers participated in the study. Regarding criteria used for TA-TMA diagnosis, centers reported as follows: 41% of centers used the International Working Group (IWG) criteria, 41% used "overall TA-TMA" criteria and 18% used physician's decision. The threshold of schistocytes to establish TA-TMA diagnosis in the participating centers was significantly associated with morphological results of test cases evaluations (p = 0.002). The mean number of schistocytes reported from blood slide analyses were 4.3 ± 4.5% for TA-TMA cases (range 0-19.6%, coefficient of variation (CV) 0.7) and 1.3 ± 1.6% for control cases (range 0-8.3%, CV 0.8). Half of the centers reported schistocyte levels below 4% for 7/10 TA-TMA cases. The intracenter variability was low, indicating differences in the institutional practices of morphological evaluation. In conclusion, the survey identified the need for the standardization of TA-TMA morphological diagnosis.

Altered erythropoiesis and decreased number of erythrocytes in children with neuroblastoma.

Neuroblastoma (NB) is a pediatric tumor presenting at diagnosis either as localized or metastatic disease, which mainly involves the bone marrow (BM). The physical occupancy of BM space by metastatic NB cells has been held responsible for impairment of BM function. Here, we investigated whether localized or metastatic NB may alter hematopoietic lineages' maturation and release of mature cells in the periphery, through gene expression profiling, analysis of BM smears, cell blood count and flow cytometry analysis. Gene ontology and disease-associated analysis of the genes significantly under-expressed in BM resident cells from children with localized and metastatic NB, as compared to healthy children, indicated anemia, blood group antigens, and heme and porphyrin biosynthesis as major functional annotation clusters. Accordingly, in children with NB there was a selective impairment of erythrocyte maturation at the ortho-chromic stage that resulted in reduced erythrocyte count in the periphery, regardless of the presence of metastatic cells in the BM. By considering all NB patients, low erythrocyte count at diagnosis associated with worse survival. Moreover, in the subset of metastatic patients, low erythrocyte count, hemoglobin and hematocrit and high red cell distribution width at follow-up also associated with worse outcome. These observations provide an alternative model to the tenet that infiltrating cells inhibit BM functions due to physical occupancy of space and may open a new area of research in NB to understand the mechanism(s) responsible for such selective impairment.

V(H)3 and V(H)6 immunoglobulin M repertoire reconstitution after hematopoietic stem-cell transplantation in children.

BACKGROUND: Immune reconstitution after hematopoietic stem-cell transplantation (HSCT) occurs gradually. Thus, a variable period of immunodeficiency may be present, leading to immunomediated complications, such as graft-versus-host disease (GVHD) and opportunistic infections. METHODS: To better understand the kinetics of B-cell repertoire reconstitution in children, 49 pediatric patients were analyzed before and after transplantation by immunoglobulin (Ig) HCDR3 fingerprinting, which is a molecular technique that analyzes one of the hypervariable segments of the Ig heavy chain, which provides the amino acid residues that are essential to interact with antigens. RESULTS: In healthy donors, the CDR3 fingerprinting profile shows 16 to 20 bands, and each band corresponds to a particular length of CDR3. This situation is considered polyclonal. Patients analyzed just after transplantation show strong oligoclonality, because only a few CDR3 bands are detected within the first 3 to 6 months. CONCLUSIONS: The authors' data show a significant lag in diversification of the B-cell repertoire, which reaches the polyclonal situation of normal healthy donors approximately 6 months after HSCT. This period may vary depending on the type of transplant (autologous vs. allogeneic) and on the immunosuppressive therapy related to GVHD.

Monosomal complex karyotype in pediatric mixed phenotype acute leukemia.

We report on a pediatric case of mixed phenotype acute leukemia with myeloid and T-lymphoid differentiation, a single myeloblastic cell population, and a monosomal complex karyotype. The patient, a 5-year-old girl, responded to acute myeloid leukemia-oriented therapy that was decided based on the morphological appearance of blast cells. In this study, we analyzed the patient's peculiar chromosomal abnormalities, as evaluated by array comparative genomic hybridization in combination with multicolor fluorescence in situ hybridization and cytogenetic analyses.

Unrelated HSCT in an adolescent affected by congenital erythropoietic porphyria.

CEP is a rare inborn error of porphyrin-heme synthesis. Clinical manifestations can range from mild to severe and include erythrodontia, reddish-colored urine, and hemolytic anemia that can be mild or severe and may result in splenomegaly. Completely avoiding exposure to the sun is crucial. Attempts to reduce erythropoiesis and to lower circulating porphyrin levels by means of erythrocyte transfusions have been successful in reducing the expression of the disease. However, the complications of a chronic transfusion regimen are potentially severe. Successful bone marrow transplantation has been reported in CEP. We report a case of successful bone marrow transplantation and prolonged follow-up in an adolescent CEP patient.

Three consecutive related bone marrow transplants for juvenile myelomonocytic leukaemia.

Allogeneic haematopoietic stem cell transplantation (allo-HSCT) is the only cure for juvenile myelomonocytic leukaemia (JMML), but relapse remains the major cause of failure. A second transplant may be considered a way to induce the graft vs. leukaemia effect in patients who relapse after their first HSCT. We describe a 7-month-old girl with JMML who relapsed after a first, related allo-HSCT, and who again relapsed 8 months after the second transplant, despite discontinuation of immusuppressive therapy. She underwent a third allogeneic transplant from another related donor. At the time of this report the patient is in complete remission 26 months after the third transplant. We suggest that a third allo-HSCT may be taken into consideration for JMML patients who experience relapse, even after two previous transplants.