The impact of comparative genomic hybridization/single-nucleotide polymorphism microarray in risk stratification of pediatric acute lymphoblastic leukemia.

BACKGROUND: The objective of this study is to assess the concordance and added value of combined comparative genomic hybridization plus single-nucleotide polymorphism microarray (CGH/SNP) analyses in pediatric acute lymphoblastic leukemia (ALL) risk stratification compared to conventional cytogenetic methods. PROCEDURE: This is a retrospective study that included patients aged 1-18 years diagnosed with de novo ALL at Sainte-Justine Hospital between 2016 and 2021. Results from conventional cytogenetic and molecular analyses were collected and compared to those of CGH/SNP. RESULTS: A total of 135 ALL patients were included. Sample failures or non-diagnostic analyses occurred in 17.8% cases with G-banding karyotypes versus 1.5% cases with CGH/SNP. The mean turnaround time for results was significantly faster for CGH/SNP than karyotype with 5.8 versus 10.7 days, respectively. The comparison of ploidy assessment by CGH/SNP and G-banding karyotype showed strong concordance (r = .82, p < .001, r2 = .68). Furthermore, G-banding karyotype did not detect additional clinically relevant aberrations that were missed by the combined analysis of CGH/SNP and fluorescence in situ hybridization. The most common gene alterations detected by CGH/SNP were deletions involving CDKN2A (35.8%), ETV6 (31.3%), CDKN2B (28.4%), PAX5 (20.1%), IKZF1 (12.7%), and copy-neutral loss of heterozygosity (CN-LOH) of 9p (9.0%). Among these, only ETV6 deletion was found to have a significant prognostic impact with superior event-free survival in both univariate and multivariate analyses (adjusted hazard ratio 0.08, 95% confidence interval: 0.01-0.50, p = .02). CONCLUSION: CGH/SNP provided faster, reliable, and highly concordant results than those obtained by conventional cytogenetics. CGH/SNP identified recurrent gene deletions in pediatric ALL, of which ETV6 deletion conferred a favorable prognosis.

Gray Zone Lymphoma Arising in the Neck of a Teenager With a Germline Mutation in TP53.

Gray zone lymphoma is an aggressive disease for which appropriate management is still debated. We report a 15-year-old girl with a cervical mass, an enlarged ipsilateral tonsil, and anemia. Both sites showed hypermetabolism on F18-FG positron emission tomography/CT. Surgical resection was diagnostic of Epstein-Barr virus-negative gray zone lymphoma cervical and tonsillar involvement. No abnormality was found in cytogenetic analysis on tumor cells. However, exome sequencing in peripheral blood DNA revealed a germline mutation in TP53. Complete response was achieved after surgery and 6 cycles of rituximab with dose-adjusted etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin regimen.

Molecular Profiling of Hard-to-Treat Childhood and Adolescent Cancers.

Importance: Little progress in pediatric cancer treatment has been noted in the past decade, urging the development of novel therapeutic strategies for adolescents and children with hard-to-treat cancers. Use of comprehensive molecular profiling in the clinical management of children and adolescents with cancer appears a suitable approach to improve patient care and outcomes, particularly for hard-to-treat cases. Objective: To assess the feasibility of identifying potentially actionable mutations using next-generation sequencing-based assays in a clinically relevant time frame. Design, Setting, and Participants: This diagnostic study reports the results of the TRICEPS study, a prospective genome sequencing study conducted in Québec, Canada. Participants, aged 18 years or younger at diagnosis, with refractory or relapsed childhood and adolescent cancers were enrolled from April 2014 through January 2018. Whole-exome sequencing (WES) of matched tumor normal samples and RNA sequencing of tumor were performed to identify single-nucleotide variants, fusion transcripts, differential gene expression, and copy number alterations. Results reviewed by a team of experts were further annotated, synthesized into a report, and subsequently discussed in a multidisciplinary molecular tumor board. Main Outcomes and Measures: Molecular profiling of pediatric patients with hard-to-treat cancer, identification of actionable and targetable alteration needed for the management of these patients, and proposition of targeted and personalized novel therapeutic strategies. Results: A total of 84 patients with hard-to-treat cancers were included in the analysis. These patients had a mean (range) age of 10.1 (1-21) years and a similar proportion of male (45 [54%]) and female (39 [46%]). Sixty-two patients (74%) had suitable tissues for multimodal molecular profiling (WES and RNA sequencing). The process from DNA or RNA isolation to genomic sequencing and data analysis steps took a median (range) of 24 (4-41) days. Potentially actionable alterations were identified in 54 of 62 patients (87%). Actions were taken in 22 of 54 patients (41%), and 18 (33%) either were on a second or third line of treatment, were in remission, or had stable disease and thus no actions were taken. Conclusions and Relevance: Incorporating genomic sequencing into the management of hard-to-treat childhood and adolescent cancers appeared feasible; molecular profiling may enable the identification of potentially actionable alterations with clinical implications for most patients, including targeted therapy and clinically relevant information of diagnostic, prognostic, and monitoring significance.

A B-cell lymphoma-associated chromosomal translocation in a progressive transformation of germinal center.

Progressive transformation of germinal center (PTGC) is a pattern of lymph node reactive hyperplasia. It can also be the predominant pattern in a hyperplastic lymph node known as florid PTGC. It is characterized histologically by the expansion of the mantle zone lymphocytes into both the adjacent sinusoids and germinal centers. The lymphocytes destroying the germinal centers are predominantly B cells, with a minor population of T cells. Morphologically, it can be confused with nodular lymphocyte-predominant Hodgkin disease (NLPHD) because of its nodular pattern and because of the presence of large cells that can be incorrectly identified as lymphocytic and histiocytic cells. A relationship between PTGC and NLPHD remains unclear, and many authors have suggested that PTGC can represent a precursor lesion of NLPHD. Here we report the first karyotype obtained in PTGC, in a 12-year-old boy. It shows a t(3;22)(q27;q11) translocation, probably involving the BCL6 gene. This translocation has previously been described in diffuse large B-cell lymphomas and in NLPHD with BCL6 rearrangement. This finding offers an insight into a possible tumorigenic pathway from PTGC to NLPHD. Further studies will be required to confirm this hypothesis.

Fetomaternal hemorrhage during external cephalic version.

OBJECTIVE: To estimate the frequency and volume of fetomaternal hemorrhage during external cephalic version for term breech singleton fetuses and to identify risk factors involved with this complication. METHODS: A prospective observational study was performed including all patients undergoing a trial of external cephalic version for a breech presentation of at least 36 weeks of gestation between 1987 and 2001 in our center. A search for fetal erythrocytes using the standard Kleihauer-Betke test was obtained before and after each external cephalic version. The frequency and volume of fetomaternal hemorrhage were calculated. Putative risk factors for fetomaternal hemorrhage were evaluated by chi(2) test and Mann-Whitney U test. RESULTS: A Kleihauer-Betke test result was available before and after 1,311 trials of external cephalic version. The Kleihauer-Betke test was positive in 67 (5.1%) before the procedure. Of the 1,244 women with a negative Kleihauer-Betke test before external cephalic version, 30 (2.4%) had a positive Kleihauer-Betke test after the procedure. Ten (0.8%) had an estimated fetomaternal hemorrhage greater than 1 mL, and one (0.08%) had an estimated fetomaternal hemorrhage greater than 30 mL. The risk of fetomaternal hemorrhage was not influenced by parity, gestational age, body mass index, number of attempts at version, placental location, or amniotic fluid index. CONCLUSION: The risk of detectable fetomaternal hemorrhage during external cephalic version was 2.4%, with fetomaternal hemorrhage more than 30 mL in less than 0.1% of cases. These data suggest that the performance of a Kleihauer-Betke test is unwarranted in uneventful external cephalic version and that in Rh-negative women, no further Rh immune globulin is necessary other than the routine 300-microgram dose at 28 weeks of gestation and postpartum. LEVEL OF EVIDENCE: II.

Glanzmann thrombasthenia in an Oldenbourg filly.

An 18-month-old Oldenbourg filly was presented with a bleeding diathesis. Laboratory testing included platelet count, gingival bleeding time, prothrombin time (PT), activated partial thromboplastin time (aPTT), von Willebrand factor (vWf) antigen, clottable fibrinogen, clot retraction time, PFA-100 closure time, platelet aggregometry (on platelet-rich plasma), and thrombelastography (TEG). TEG was performed by using kaolin and tissue factor as coagulation activators. Expression of the platelet receptor for fibrinogen was assessed by flow cytometry by using anti CD41 (alpha(IIb) or glycoprotein IIb)/CD61 (beta(III) or glycoprotein IIIa) and anti-CD41 antibodies. Abnormal laboratory findings included prolonged oral mucosal bleeding time (>12 hours), prolonged closure time with collagen/ADP (>300 seconds), and absence of clot retraction after 60 minutes. TEG reaction times were similar with kaolin and tissue factor in the patient and a control horse. However, maximum amplitudes in the patient were decreased with both kaolin (43.7 mm; control, 63.9 mm) and tissue factor (37.7 mm; control, 57.8 mm). Platelet aggregation responses to ADP and collagen were profoundly reduced in the affected horse compared with a control. Flow cytometry showed an absence of CD41 and decreased expression of CD41/CD61-reacting antigen on the patient's platelets compared with those from a control horse. The laboratory findings supported a diagnosis of Glanzmann thrombasthenia, likely caused by a mutation in the gene encoding the GPIIb subunit.

Polymorphisms in genes encoding drugs and xenobiotic metabolizing enzymes, DNA repair enzymes, and response to treatment of childhood acute lymphoblastic leukemia.

PURPOSE: The most common childhood malignancy, acute lymphoblastic leukemia (ALL), remains the leading cause of cancer-related death in children because of resistant cases in which underlying predisposing factors are poorly understood. The interindividual variation in the activity of xenobiotic metabolizing enzymes that modify individual somatic mutation burden in the context of environmental exposure was shown to modify susceptibility to childhood ALL. Variable DNA repair capacity may further modulate induced DNA lesions. Similarly, differential capacity of ALL patients to process carcinogens and chemotherapeutic drugs could both modify an individual's risk of recurrent malignancy and response to therapy. EXPERIMENTAL DESIGN: We investigated the relationship between the risk of relapse in ALL patients and functional polymorphisms in genes encoding carcinogen-metabolizing enzymes, including CYP1A1, CYP2D6, CYP2E1, MPO, GSTM1, GSTT1, GSTP1, NAT1, NAT2, NQO1, as well as DNA-repair enzymes hMLH1, hMSH3, XRCC1, XPF, and APE. Our study included 320 children with ALL, of which 68 relapsed or died because of this disease within 5 years of follow-up. RESULTS: Among children of the latter group, we found that carriers of CYP1A1*2A and NQO1*2 variants had worse disease prognosis according to Kaplan-Meier (P = 0.003) and Cox regression (P

Clinical and genetic analysis of unclassifiable inherited bone marrow failure syndromes.

OBJECTIVE: Unclassified inherited bone marrow failure syndromes are a heterogeneous group of genetic disorders that represent either new syndromes or atypical clinical courses of known inherited bone marrow failure syndromes. The relative prevalence of the unclassified inherited bone marrow failure syndromes and their characteristics and the clinical and economic challenges that they create have never been studied. METHODS: We analyzed cases of inherited bone marrow failure syndrome in the Canadian Inherited Marrow Failure Registry that were deemed unclassifiable at study entry. RESULTS: From October 2001 to March 2006, 39 of the 162 patients enrolled in the Canadian Inherited Marrow Failure Registry were registered as having unclassified inherited bone marrow failure syndromes. These patients presented at a significantly older age (median: 9 months) than the patients with classified inherited bone marrow failure syndrome (median: 1 month) and had substantial variation in the clinical presentations. The hematologic phenotype, however, was similar to the classified inherited bone marrow failure syndromes and included single- or multiple-lineage cytopenia, severe aplastic anemia, myelodysplasia, and malignancy. Grouping patients according to the affected blood cell lineage(s) and to the presence of associated physical malformations was not always sufficient to characterize a condition, because affected members from several families fit into different phenotypic groups. Compared with the classified inherited bone marrow failure syndromes, the patients with unclassified inherited bone marrow failure syndromes had 3.2 more specific diagnostic tests at 4.5 times higher cost per evaluated patient to attempt to categorize their syndrome. At last follow-up, only 20% of the unclassified inherited bone marrow failure syndromes were ultimately diagnosed with a specific syndrome on the basis of the development of new clinical findings or positive genetic tests. CONCLUSIONS: Unclassified inherited bone marrow failure syndromes are relatively common among the inherited bone marrow failure syndromes and present a major diagnostic and therapeutic dilemma.

Complications of apheresis in children.

BACKGROUND: Although the frequency of complications in adults undergoing therapeutic apheresis is low, there are little data in children. STUDY DESIGN AND METHODS: A retrospective study of 186 children who had undergone a total of 1632 apheresis procedures between 1994 and 2002 was conducted. Adverse reactions were prospectively documented. The procedures were plasma exchange (67%), hematopoietic progenitor cell collection (18%), red blood cell exchange (6.9%), leukodepletion (0.7%), and plasma exchange with immunoadsorption (6.7%). RESULTS: Adverse reactions, most minor, were reported in 55 percent of procedures in 82 percent of patients. The most frequent complications, per procedure and per patient during an entire course of therapy, were hypotension (14 and 48.4%), hypotension requiring fluid bolus (4.8 and 26.9%), symptomatic hypocalcemia (9.7 and 28.5%), allergic reactions (4.4 and 5.9%), catheter-related thrombosis (1.7 and 12.4%), catheter-related infection (2.1 and 16.1%), and severe anemia (hemoglobin [Hb] level, <7 g/dL; 2.5 and 17.2%). There were two deaths (1% of patients). Risk factors for complications by multivariate analysis were lower body weight, lower preapheresis Hb level, apheresis in a critical care unit, and number of procedures per patient. The 55 percent incidence of complications per procedure in our pediatric cohort is much higher than the 4.3 to 28 percent incidence reported in adults. The excess of adverse reactions in children are mostly related to citrate toxicity, higher relative vascular volume shifts, and the need for vascular access. CONCLUSION: Pediatric apheresis presents unique challenges and is associated with higher complication rate compared to adults. It is recommended that this procedure be performed in specialized centers.

Assessment of cord blood unit characteristics on the day of transplant: comparison with data issued by cord blood banks.

BACKGROUND: Selection of a cord blood (CB) unit for allogeneic transplantation relies on graft characterization results provided by cord blood banks (CBBs). The goal was to compare the graft characterization results obtained upon thawing and washing to those provided by CBBs at selection. STUDY DESIGN AND METHODS: With tests that assess CB graft characteristics known to impact engraftment, CB units have been analyzed after thaw and before infusion. Our results were compared to data provided by CBBs to determine the impact on engraftment and assess how CBB-supplied information can affect future CB unit selection. RESULTS: Variability was noted as to the type of information provided by the different CBBs. Also, variability was found between the information provided by CBBs and the graft characterization results obtained upon thawing and washing. In some cases, CB measures known to be predictive of engraftment were found much lower than reported by CBBs. Only the total nucleated cell count, which is the main CB graft selection criterion besides HLA matching, correlated favorably. CONCLUSIONS: Our data reveal a high degree of variability in graft characteristics provided by CBBs and often poor correlation with results obtained on thawed and washed CB units. We suggest that standardized laboratory procedures aimed at graft characterization should be used by both CBBs and transplant centers to avoid unacceptable discrepancies.

Characterization of cord blood natural killer cells: implications for transplantation and neonatal infections.

The role of natural killer (NK) cells in hematopoietic stem cell transplantation and in the control of neonatal infections is not yet clear. Donor-versus-recipient NK cell alloreactivity was found to improve outcome in some settings of hematopoietic stem cell transplantation. We hypothesized that the role of NK cells in cord blood (CB) transplantation and neonatal infections may depend on CB NK cell maturation stage. We therefore analyzed the expression of NK cell differentiation/phenotypic markers in human CB, as well as functional properties of purified CB NK cells. CD8 and CD57 expression was lower in CB than in adult NK cells. However, the expression of other differentiation markers was similar, as was cell surface density of CD56, the percentage of late NK cell precursors, interferon-gamma production, and the proliferative response of purified NK cells to IL-2. Spontaneous cytotoxic activity of purified CB NK cells against NK-sensitive targets was low but reached adult levels after treatment with IL-15. Expression of perforin and granzyme B was higher in CB NK cells (90 versus 58% and 86 versus 69%, respectively). intercellular adhesion molecule-1 and CD161 expression was lower in CB. Surprising, fewer CB NK cells expressed L-selectin, a marker of immature NK cells. Taken together, our results suggest that CB NK cells are phenotypically and functionally mature.

Second induction in pediatric patients with recurrent acute lymphoid leukemia using DFCI-ALL protocols.

Between 15% and 30% of children with acute lymphoblastic leukemia (ALL) experience disease recurrence. With the possible exception of patients presenting with late isolated extramedullary relapse, induction of second complete remission (CR) is employed as a stepping stone to allogeneic hematopoietic stem cell transplantation (HSCT). The authors report their institutional experience in the management of children with recurrent ALL using the Dana Farber Cancer Institute (DFCI) ALL protocol in patients treated initially with that same protocol. Successful reinduction was followed by allogeneic HSCT when possible. Between April 1986 and May 2003, 34 patients with recurrent ALL, treated at initial diagnosis with DFCI-ALL protocol therapy, were given the same protocol as repeat induction chemotherapy. The median age was 4.6 years at diagnosis and 7.1 years at recurrence. Median duration of CR1 was 30.3 months. Second CR was obtained in 29 (85%) patients. Twenty went on to allogeneic HSCT; 10 of them currently remain in CR. Two additional patients treated with chemotherapy without HSCT are also in continuous CR2. Overall, 13 (38%) of the 34 patients are alive with a median follow-up of 105 months. There were no toxic deaths due to the reinduction therapy. One child died of cardiac failure after autologous HSCT. The treatment of children with recurrent ALL using the DFCI-ALL protocol induction regimen after initial use of the same protocol is associated with a high rate of second CR with no excess toxicity. However, the overall prognosis in these patients remains unsatisfactory and needs to be improved.