Despite attempts to improve the definitions of ambiguous lineage leukemia (ALAL) during the last 2 decades, general therapy recommendations are missing. Herein, we report a large cohort of children ...with ALAL and propose a treatment strategy. A retrospective multinational study (International Berlin-Frankfurt-Münster Study of Leukemias of Ambiguous Lineage iBFM-AMBI2012) of 233 cases of pediatric ALAL patients is presented. Survival statistics were used to compare the prognosis of subsets and types of treatment. Five-year event-free survival (EFS) of patients with acute lymphoblastic leukemia (ALL)–type primary therapy (80% ± 4%) was superior to that of children who received acute myeloid leukemia (AML)–type or combined-type treatment (36% ± 7.2% and 50% ± 12%, respectively). When ALL- or AML-specific gene fusions were excluded, 5-year EFS of CD19+ leukemia was 83% ± 5.3% on ALL-type primary treatment compared with 0% ± 0% and 28% ± 14% on AML-type and combined-type primary treatment, respectively. Superiority of ALL-type treatment was documented in single-population mixed phenotype ALAL (using World Health Organization and/or European Group for Immunophenotyping of Leukemia definitions) and bilineal ALAL. Treatment with ALL-type protocols is recommended for the majority of pediatric patients with ALAL, including cases with CD19+ ALAL. AML-type treatment is preferred in a minority of ALAL cases with CD19− and no other lymphoid features. No overall benefit of transplantation was documented, and it could be introduced in some patients with a poor response to treatment. As no clear indicator was found for a change in treatment type, this is to be considered only in cases with ≥5% blasts after remission induction. The results provide a basis for a prospective trial.
•The largest cohort of ambiguous leukemias to date reveals a better prognosis of children who started on lymphoid-directed treatment.•Myeloid-type primary treatment correlated with dismal outcomes in CD19+ leukemias.
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Optimal discrimination between leukemic blasts and normal B-cell precursors (BCP) is critical for treatment monitoring in BCP acute lymphoblastic leukemia (ALL); thus identification of markers ...differentially expressed on normal BCP and leukemic blasts is required.
Multicenter analysis of CD73, CD86 and CD304 expression levels was performed in 282 pediatric BCP-ALL patients vs. normal bone marrow BCP, using normalized median fluorescence intensity (nMFI) values.
CD73 was expressed at abnormally higher levels (vs. pooled normal BCP) at diagnosis in 71/108 BCP-ALL patients (66%), whereas CD304 and CD86 in 119/202 (59%) and 58/100 (58%) patients, respectively. Expression of CD304 was detected at similar percentages in common-ALL and pre-B-ALL, while found at significantly lower frequencies in pro-B-ALL. A significant association (p = 0.009) was found between CD304 expression and the presence of the ETV6-RUNX1 fusion gene. In contrast, CD304 showed an inverse association with MLL gene rearrangements (p = 0.01). The expression levels of CD73, CD86 and CD304 at day 15 after starting therapy (MRD15) were stable or higher than at diagnosis in 35/37 (95%), 40/56 (71%) and 19/41 (46%) cases investigated, respectively. This was also associated with an increased mean nMFI at MRD15 vs. diagnosis of +24 and +3 nMFI units for CD73 and CD86, respectively. In addition, gain of expression of CD73 and CD86 at MRD15 for cases that were originally negative for these markers at diagnosis was observed in 16% and 18% of cases, respectively. Of note, CD304 remained aberrantly positive in 63% of patients, despite its levels of expression decreased at follow-up in 54% of cases.
Here we show that CD73, CD86 and CD304 are aberrantly (over)expressed in a substantial percentage of BCP-ALL patients and that their expression profile remains relatively stable early after starting therapy, supporting their potential contribution to improved MRD analysis by flow cytometry.
•An original method of determination of antigen expression was used.•Increased expression of CD304 is associated with specific subtypes of BCP-ALL.•The utility of CD73, CD86 and CD304 markers for MRD monitoring in BCP-ALL was confirmed.•Of the 3 markers evaluated, CD73 was the most stable at early follow-up time points.
Familial hemophagocytic lymphohistiocytosis (FHL) is a genetically determined hyperinflammatory syndrome caused by uncontrolled immune response mediated by T-lymphocytes, natural killer (NK) cells, ...and macrophages. STXBP2 mutations have recently been associated with FHL5. To better characterize the genetic and clinical spectrum of FHL5, we analyzed a cohort of 185 patients with suspected FHL for mutations in STXBP2. We detected biallelic mutations in 37 patients from 28 families of various ethnic origins. Missense mutations and mutations affecting 1 of the exon 15 splice sites were the predominant changes detectable in this cohort. Patients with exon 15 splice-site mutations (n = 13) developed clinical manifestations significantly later than patients with other mutations (median age, 4.1 year vs 2 months) and showed less severe impairment of degranulation and cytotoxic function of NK cells and CTLs. Patients with FHL5 showed several atypical features, including sensorineural hearing deficit, abnormal bleeding, and, most frequently, severe diarrhea that was only present in early-onset disease. In conclusion, we report the largest cohort of patients with FHL5 so far, describe an extended disease spectrum, and demonstrate for the first time a clear genotype-phenotype correlation.
•Pediatric CD371-positive B-cell precursor acute lymphoblastic leukemia shows transient lineage switch and slow early response to treatment.•Accurate immunophenotypic identification of lineage switch ...is mandatory to properly assess MRD by flow cytometry.
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In the effort to improve immunophenotyping and minimal residual disease (MRD) assessment in acute lymphoblastic leukemia (ALL), the international Berlin-Frankfurt-Münster (iBFM) Flow Network introduced the myelomonocytic marker CD371 for a large prospective characterization with a long follow-up. In the present study, we aimed to investigate the clinical and biological features of CD371-positive (CD371pos) pediatric B-cell precursor ALL (BCP-ALL). From June 2014 to February 2017, 1812 pediatric patients with newly diagnosed BCP-ALLs enrolled in trial AIEOP-BFM ALL 2009 were evaluated as part of either a screening (n = 843, Italian centers) or validation cohort (n = 969, other iBFM centers). Laboratory assessment at diagnosis consisted of morphological, immunophenotypic, and genetic analysis. Response assessment relied on morphology, multiparametric flow cytometry (MFC), and polymerase chain reaction (PCR)-MRD. At diagnosis, 160 of 1812 (8.8%) BCP-ALLs were CD371pos. This correlated with older age, lower ETV6::RUNX1 frequency, immunophenotypic immaturity (all P < .001), and strong expression of CD34 and of CD45 (P < .05). During induction therapy, CD371pos BCP-ALLs showed a transient myelomonocytic switch (mm-SW: up to 65.4% of samples at day 15) and an inferior response to chemotherapy (slow early response, P < .001). However, the 5-year event-free survival was 88.3%. Among 420 patients from the validation cohort, 27 of 28 (96.4%) cases positive for DUX4-fusions were CD371pos. In conclusion, in the largest pediatric cohort, CD371 is the most sensitive marker of transient mm-SW, whose recognition is essential for proper MFC MRD assessment. CD371pos is associated to poor early treatment response, although a good outcome can be reached after MRD-based ALL-related therapies.
Hepatitis-associated aplastic anemia (HAAA) is a rare variant of bone marrow (BM) failure that is typically diagnosed 2-3 months after an acute attack of hepatitis in patients that are seronegative ...for known hepatitis viruses. Although very little is known about its etiology, an autoimmune mechanism is presumed based on clinical response to immunosuppressive therapy (IST) and reports of oligoclonal T-lymphocyte expansion and skewing of CDR3 region length. However due to the rareness of this disease, the published evidence is still limited.
We analyzed 18 pediatric patients consecutively diagnosed with HAAA in the Czech Republic between 2004-2019. Based on BM biopsy examination, 14 patients had aplastic anemia (AA) and interestingly 4 patients were histologically consistent with refractory cytopenia of childhood (RCC). Median age of these patients was 6.6 years (range 1-18) with a male to female ratio of 2:1. The prevalence of HAAA in our cohort of pediatric patients with BM failure (n=107; excluding patients with known genetic cause) was 17%. The presence of a paroxysmal nocturnal hemoglobinuria clone was excluded and cytogenetic evaluation showed no abnormalities.
We examined both the diagnostic BM and peripheral blood by flow cytometry in all patients (with the exception of one peripheral blood sample) and performed deep immunophenotyping of T cells in 7 of them. Ten patients were selected for T cell receptor beta (TRB) repertoire and whole-exome sequencing (WES) based on the availability of DNA samples isolated from the BM before the start of the treatment. TRB sequencing was performed following the SOP developed by the EuroClonality-NGS working group with DNA input normalized to the equivalent of 20 000 CD3+ cells per sample based on flow cytometry data. Bioinformatic analysis was performed with ARResT/Interrogate. WES was performed on Illumina NextSeq 500 and libraries prepared using the Agilent SureSelectXT Human All Exon V6+UTRs kit. Vertebrate virus detection was performed using the VirCapSeq-VERT probe capture enrichment set and massive parallel sequencing from 8 BM DNA samples taken at the time of diagnosis.
We found significant activation of CD8 positive T cells based on the expression of HLA-DR compared to non-HAAA RCC and AA patients (n=20 and n=21 respectively, Mann-Whitney p<0.0001). Three out of 7 patients with deep T cell immunophenotype available showed a decrease of naive CD27pos45RAposCD8pos T cells. From TRB repertoire analysis we were able to identify a total of 5 private immunodominant clones in 3 patients with relative abundance of up to 11% from all productive TRB sequences. These clones were not found in any previous publications or clonotype databases based on their CDR3 sequence. We detected 5 clones shared by 3 or more patients; these were previously published in other cohorts and are not specific for HAAA. WES analysis did not reveal any previously published variant in genes associated with immune dysregulation or immunodeficiency, or any candidate variant for functional validation. No relevant vertebrate virus signal was detected in the 8 samples.
Out of all 18 patients, 3 patients underwent MSD-HSCT without preceding immunotherapy. Fifteen patients were treated according to standard European Working Group on MDS/SAA protocols with anti-thymocyte globulin (horse n=6; rabbit n=9), corticosteroids and cyclosporin A. In total, 7 patients reached complete remission on IST by day 120 and 7 patients with insufficient response underwent MUD-HSCT. One patient died shortly after MUD-HSCT because of toxicity. We did not observe any correlation of T cell populations at diagnosis or day 120 of IST (activated and exhausted T cells) and clinical response. Similarly, the presence of an immunodominant clone or oligoclonal TRB repertoire with lower diversity at diagnosis had no significant impact on the outcome of these patients in our cohort.
In conclusion, we present the biggest cohort of pediatric patients with HAAA analyzed by next-generation sequencing and flow cytometry so far. Even though our results show significantly increased activation of CD8 positive T-cells and presence of expanded clones, we did not confirm that these factors would be useful and significant for the prediction of treatment outcome. Large number of our patients did not fully respond to IST, requiring HSCT.
Supported by AZV 18-07-00430, 16-32568A and PRIMUS/17/MED/11.
No relevant conflicts of interest to declare.
GATA-2 deficiency was recently described as common cause of overlapping syndromes of immunodeficiency, lymphedema, familiar myelodysplastic syndrome or acute myeloid leukemia. The aim of our study ...was to analyze bone marrow and peripheral blood samples of children with myelodysplastic syndrome or aplastic anemia to define prevalence of the GATA2 mutation and to assess whether mutations in GATA-2 transcription factor exhibit specific immunophenotypic features. The prevalence of a GATA2 mutation in a consecutively diagnosed cohort of children was 14% in advanced forms of myelodysplastic syndrome (refractory anemia with excess blasts, refractory anemia with excess blasts in transformation, and myelodysplasia-related acute myeloid leukemia), 17% in refractory cytopenia of childhood, and 0% in aplastic anemia. In GATA-2-deficient cases, we found the most profound B-cell lymphopenia, including its progenitors in blood and bone marrow, which correlated with significantly diminished intronRSS-Kde recombination excision circles in comparison to other myelodysplastic syndrome/aplastic anemia cases. The other typical features of GATA-2 deficiency (monocytopenia and natural killer cell lymphopenia) were less discriminative. In conclusion, we suggest screening for GATA2 mutations in pediatric myelodysplastic syndrome, preferentially in patients with impaired B-cell homeostasis in bone marrow and peripheral blood (low number of progenitors, intronRSS-Kde recombination excision circles and naïve cells).
Acute lymphoblastic leukemia treatment leads to elimination of blasts and stepwise regeneration of normal hematopoiesis. Several studies identified prognostic relevance of minimal residual disease ...(MRD) in bone marrow (BM) before achieving complete remission (Giuseppe Basso et al., J Clin Oncol, 2009). Crucial question is how to assess BM quality at day 15 (d15) of ALL BFM protocols. In ALL BFM 2009 protocol good quality sample is defined as containing more than 2% erythroid precursors (EP) of nucleated cells. EP were defined as CD19neg(orCD7neg)CD45neg.
Two pt cohorts were included in the study. First cohort (Coh2000) consisted of pts treated by AIEOP BFM ALL 2000, n=196 (177 BCP ALL, 19 T ALL, median follow-up 5.4 yrs, range 0.025-10). AIEOP BFM ALL 2000 study was a PCR MRD based protocol (assessment at day d33 and d78) and flow cytometric MRD (FC MRD) was assessed only on research basis at d15. Second cohort (Coh2009) consisted of pts treated by AIEOP BFM ALL 2009, n=331 (292 BCP ALL, 39 T ALL, median follow up 4.8 yrs; range 0.0027-7.6). In Coh2009, both PCR MRD (d33, d78) and FC MRD d15 were used for risk stratification.
We asked following questions:What is the specificity and viability of EP defined by CD45 negativity? Is a definition based on bright expression CD71 more specific?Is the amount of EP different between B and T ALLs and between risk groups defined by FC at d15? What is the overall frequency of low EP at d15?What is the relationship between amount of EP and FC MRD at d15?Is there any prognostic relevance of low EP?
Results:Population of EP was selected based on negativity of CD45 and a lineage marker (CD19 or CD7) among nucleated cells, which were defined as positive by a SYTO nucleic fluorescent dye. We found a high amount of non-viable cells defined by 4′,6-diamidino-2-phenylindole (DAPI) positivity (6.5-96%, median 55%). When we added bright CD71 into the EP definition (EP CD71++), the percentage of DAPI positivity was significantly lower (0-66%, median 9%) (p<0.0001 in both cohorts).There is no difference in amount of EP at d15 between B and T ALL in either of the cohorts. The treatment reduction in SR pts (FC MRD d15<0.1%) was in Coh2009 used only in BCP ALL and we focused in further analyses on BCP ALLs. Overall, the EP were below 2% in 16% and 18% in Coh2000 and Coh2009, respectively. Within risk groups, EP below 2% at d15 occurred more frequently in Standard Risk (SR; 27%) than in non-SR (non-SR; 12%) in both cohorts (p=0.0002). The frequency of low EP appears higher than the expected frequency of technically poor samples. Moreover, it is unlikely that quality of BM aspiration would depend on the risk group of the pt. This further supports the role of normal BM response to presence of leukemic cells on one hand and to therapy on the other one.In both cohorts we found significant positive correlation between amount of EP and FC MRD at day 15 (Coh2000 p-value= 0.0016 (R 0.23); Coh2009 p value <0.0001 (R 0.33)). The correlation was significant in BCP ALLs only (Coh2000 p value=0.008 (R 0.26); Coh2009 p value<0.0001 (R 0.39)). The same significant correlation is observed in BCP ALLs with more precisely defined population EP CD71++DAPIneg (Coh2000 p value=0.04 (R0.18); Coh2009 p<0.0001 (R 0.35)). Part of the Coh2000 was treated with prednisone and part of the pts with dexamethasone between d8 and d28, whereas the entire Coh2009 received prednisone only. However, the frequency of low EP was not different between dexamethasone and prednisone-treated pts, the correlation between FC MRD and EP was significant only in prednisone treated pts (p=0.0003, R=0.33).We focused on SR BCP ALL pts (FC MRD d15<0.1%). We did not find difference in event free survival (EFS) between pts with amount of EP below and above 2%. This result indicates that the pts with low MRD and low EP are not just pts with hemodiluted BM samples.
Conclusion: Sample quality is essential question in the assessment of MRD in BM. Although low EP may indicate poor BM aspiration quality, it may also result from other biological factors. At d15 BCP ALL, these factors include the interaction of normal BM cells with leukemia, patient's risk group, and type of corticosteroid used. EPs should be detected using an erythroid marker, such as CD71. However, new markers of BM quality, less influenced by leukemia treatment, are needed.
Supported by Ministry of Health of CR, grant nr. 15-28525A, NV18-07-00430 and NV18-03-00343; Czech Science Foundation nr. P302/12/G101.
Brüggemann:PRMA: Consultancy; Incyte: Consultancy; Pfizer: Speakers Bureau; Roche: Speakers Bureau; Affimed: Research Funding; Regeneron: Research Funding; Amgen: Consultancy, Research Funding, Speakers Bureau.
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