Recently, we described B-cell precursor acute lymphoblastic leukemia (BCP-ALL) subtype with early switch to the monocytic lineage and loss of the B-cell immunophenotype, including CD19 expression. ...Thus far, the genetic background has remained unknown. Among 726 children consecutively diagnosed with BCP-ALL, 8% patients experienced switch detectable by flow cytometry (FC). Using exome and RNA sequencing, switch was found to positively correlate with three different genetic subtypes: PAX5-P80R mutation (5 cases with switch out of 5), rearranged DUX4 (DUX4r; 30 cases of 41) and rearranged ZNF384 (ZNF384r; 4 cases of 10). Expression profiles or phenotypic patterns correlated with genotypes, but within each genotype they could not identify cases who subsequently switched. If switching was not taken into account, the B-cell-oriented FC assessment underestimated the minimal residual disease level. For patients with PAX5-P80R, a discordance between FC-determined and PCR-determined MRD was found on day 15, resulting from a rapid loss of the B-cell phenotype. Discordance on day 33 was observed in all the DUX4r, PAX5-P80R and ZNF384r subtypes. Importantly, despite the substantial phenotypic changes, possibly even challenging the appropriateness of BCP-ALL therapy, the monocytic switch was not associated with a higher incidence of relapse and poorer prognosis in patients undergoing standard ALL treatment.
Daratumumab, an anti‐CD38 antibody, is used experimentally in the treatment of relapsed acute lymphoblastic leukemia (ALL). We treated five patients suffering from relapsed ALL with daratumumab. Four ...patients had T ALL, three of whom achieved complete remission (CR) after treatment and underwent stem cell transplant (SCT). Two of them had a second relapse and died 6 and 8 months after SCT, respectively. One transplanted T ALL patient remained in CR2 15 months after relapse. In the remaining T‐ALL patient, the disease progressed under daratumumab treatment, and the patient died early after the first relapse. The B‐cell precursor ALL patient with a second CD19‐negative relapse, whose disease turned out to be resistant to the combination of daratumumab with chemotherapy, later achieved CR3 with inotuzumab ozogamicin, underwent SCT and remained in CR3. Leukemia burden should be monitored after daratumumab, and care should be taken not to misclassify leukemic cells with false negativity of surface CD38; using an antibody reacting with nondaratumumab epitopes is advantageous.
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.
Introduction:
Recently we described a subgroup of pediatric patients with B cell precursor acute lymphoblastic leukemia (BCP ALL) with switching from B to monocytic lineage in early phase of the ...therapy (Slamova et al., 2014). In a limited cohort of patients with switching ALL (swALL), we observed inferior response to treatment with discrepancy of minimal residual disease level (MRD) assessed by flow cytometry (FC) and quantitative polymerase chain reaction (qPCR) of Immunoglobulin-T cell receptor (Ig-TCR) rearrangements. In current Berlin-Frankfurt-Münster (BFM) treatment protocols, FC MRD value at day 15 (d15) and PCR MRD value at day 33 (d33) and week 12 (w12) are used for stratification. Using an extended cohort of patients with available RNA sequencing data (cohort mainly focused on B other cases or swALLs) we aimed to answer following questions:What is the genetic background of swALL? What is the frequency among swALLs of the recently described DUX4 rearranged subgroup?How do B cell oriented FC and PCR MRD correlate in standard protocol timepoints, i.e. day 8 (d8) (peripheral blood, PB) d15, d33 and w12 (bone marrow, BM) of treatment?What is the characteristic MRD response to treatment in swALL?
Results:We performed RNA sequencing in 177 patients (median age 6.1 years, range 0-18) treated by several treatment protocols (ALL BFM 95 n=5, ALL IC BFM 2002 n=14, ALL AIEOP BFM 2000 n=17, ALL AIEOP BFM 2009 n=135, Interfant n=3, ALL IC/Interfant n=2, EsPhALL n=1). In 68 patients we observed switching phenomenon by appearance of B/monocytoid population coexpressing B lineage (CD19, CD34) and monocytic lineage (CD33, CD14) markers (median 0.98%, range 0.032-38%). In non swALLs median of this population was 0.059% (range 0.0025-1.1%) and the cells did not form a clear cluster. According to RNAseq data, majority of swALL patients (n=42/68) belong to DUX4 subgroup (chi square p< 0.00001). The distribution into other molecular genetic subtypes is summarized in table 1.Correlation coefficient (Spearman) of all included samples with both available values (n=552) was 0.82 (p<0.0001), Concordance in categorization of positivity and negativity with cut-off 1e-4 was 85%. We observed worse correlation between FC and PCR MRD in patients with swALL (d8 R=0.58, d15 R=0.6, d33 R=0.36, w12 n.s.) compared to non swALL (d8 R=0.83, d15 R=0.91, d33 R=0.69, w12 R=0.37). However, concordance in swALL in categorization of positivity and negativity with cut-off 1e-4 was still ≥80% in each analyzed timepoint apart from d33 with concordance only 44% showing significant discrepancy of both methods (Figure 1a). On the contrary, concordance in non swALL was ≥87% for each analyzed timepoint (d33 with concordance 87% shown in Figure 1b). Poor correlation between B-cell oriented FC MRD and PCR MRD at d33 was also obvious when analyzed DUX4 subgroup separately (R=0.31 (p=0.04), concordance 45%).We observed significantly higher MRD in swALLs compared to non swALLs at all analyzed timepoints: d8 (p=0.0021), d15 (p=0.0088, d33 (p<0.0001) and w12 (p=0.008). Higher MRD levels were also found in DUX4 patients when compared to non DUX4 (all timepoints p<0.05). Interestingly, when compared swALL and non swALLs pts in DUX4 subgroup only, the DUX4 swALLs are those with poorer treatment response (all timepoints p<0.05). With respect to protocolar cut-off values, FC MRD at d15 was above 10% in 18/67 swALL patients (in 13/52 DUX4 patients), d33 PCR MRD was above 0.1% in 33/57 swALLs (24/44 DUX4 pts) and at w12 PCR MRD was above 0.01% in 12/55 swALLs (10/44 DUX4 pts).
Conclusions:
DUX4 subgroup is the most prevalent genetic subtype among swALLs. SwALLs and/or DUX4 subgroup have poorer treatment response at d15, d33 and w12. However, it remains to be elucidated whether poor initial treatment response is eventually reflected in treatment outcome. In majority of swALL patients B cell phenotype of blasts is preserved at day 15 enabling correct classification. Prominent discrepancy between FC and PCR MRD is present especially at d33 and development of different FC MRD strategies focused on monocytic compartment is needed.
Supported by Ministry of Health of the Czech Republic, grant nr. 15-28525A and NV18-03-00343; Czech Science Foundation nr.P302/12/G101, UNCE204012
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Brüggemann:Affimed: Research Funding; Regeneron: Research Funding; Amgen: Consultancy, Research Funding, Speakers Bureau; Roche: Speakers Bureau; Pfizer: Speakers Bureau; Incyte: Consultancy; PRMA: Consultancy. Ritgen:abbvie: Research Funding; Roche: Honoraria, Research Funding.
Switching from B to monocytic lineage is more frequent than expected (4-6% out of all pediatric B cell precursor leukemias (BCP ALLs), called swALL). SwALLs frequently contain CD2 aberrant expression ...and ERG gene deletions. Recently described subtype characterized by rearrangement of DUX4 gene overlaps with swALL. The B-to-monocytoid switching can be missed by current flow cytometry (FC) antibody panels. The search for better FC markers is of clinical importance also in the context of switching occurring under CD19 directed therapies (blinatumomab or CAR T cells). The malignant potential of switched blasts is not clear, there is an evidence that part of the patients might suffer from relapse corresponding to BCP ALL. However, the relapse corresponding to switched monocytic blasts was also observed. We performed RNA sequencing of swALL diagnostic blasts (n=31), intermediate cells co-expressing B cell and monocytic markers (n=3) and switched monocytic blasts (n=6, clonal relatedness was confirmed by identification of identical Ig-TCR rearrangements with initial BCP ALL clone), control BCP ALLs (n=50), monocytic AMLs (n=5, MLL rearranged 3, CBFb/MYH11 2) and healthy monocytes sorted from blood (n=3) to answer following questions:
a) Which genes are differentially expressed at diagnosis in swALL in comparison to non swALL?
b) Which genes are differentially expressed in switched monocytic blasts in comparison to normal monocytes?
c) Is expression profile of switched blasts similar to monocytic AML?
Results:
a) We identified following 20 most significantly differentially expressed genes at diagnosis of swALL, compared to control BCP ALL: CCNA1, ANGPT1, AR, COL9A3, CHST13, GPR37, TCERG1L, SPON2, CLEC12A (CD371)-antigen broadly expressed in AMLs and healthy monocytes, GLDC, LHFPL2, CLEC12B, PNMT, SLC35E3, SMOC2, CSRNP3, MS4A1 (CD20), STAP1, PPP1R36, FAM107B. We also looked for 20 most significantly expressed genes having CD equivalent and following molecules were identified (and therefore likely usable by FC): CD371, CD294, CD315, CD146, CD140a, CD57, CD213a1, CD49b (increased in swALLs) and CD20, CD27, CD331, CD158K, CD10, CD85h, CD130, CD268, CD185, CD200, CD247, CD114, CD49e (decreased in swALLs). Other interesting significantly expressed molecules in comparison to control BCP ALLs, are CD2 (LFA-2) and CD84 (SLAM family member 5) both belonging to Ig superfamily of molecules and FC profile is compatible with transcriptome.
b) The following genes were upregulated in comparison to healthy monocytes: IL10, NR4A3, S3ST3B1, AVPI1, PHLPP1, PLCXD1, WEE1, LARGE. Following genes were downregulated: RAB32 (RAS pathway member), CYP27A1 (high expression is typical for macrophages), NUDT16P1, HOXB2, SMPDL3A, RTN1, MN1, ZC2HC1A.
The following genes with CD equivalent were found for prospective screening: CD52 (decreased in switched blasts), CD218a (IL18R1), CD146 (MCAM), CD104 (ITGB4), CD218b (IL18RAP), CD168 (HMMR), CD358, CD227 (MUC1), CD34, CD69, CD104, CD22, CD98 (LAT1), CD125 (IL5 subunit), CD363, CD229, CD70, CD233 (SLC4A1), CD49b and CD334 (increased in switched blasts).
c) Switched monocytic blasts have distinct expression profile from healthy monocytes and monocytic AMLs. Interestingly switched monocytic blasts have higher expression of granulocyte macrophage colony stimulating factor receptor (GM CSFr, CD116) indicating higher dependence on this cytokine in comparison to monocytic AMLs.
Conclusion: SwALLs have distinct expression profile in comparison to control BCP ALLs. Expression profile at diagnosis is consistent with immunophenotype (CD19pos, CD20neg, frequently CD34pos, CD10dim and CD2pos). SwALLs tend to highly express several molecules of the C type lectin family (namely CLEC12A and CLEC12B). In comparison to healthy monocytes switched blasts retain expression of several B cell specific gene transcripts (e.g.EBF1, LARGE, CD22) and a higher expression of Ki67 corresponds to a higher proliferative capacity. SwALL represents a subset of BCP ALL with a potential to downregulate CD19 during therapy. Although our knowledge on genetic background of swALL has recently expanded, the details behind their phenotypical volatility are mostly yet unknown. Our study is the first to provide systematic insight into the swALL transcriptome and surface protein expression. Supported by AZV15-28525A, 15-30626A, 16-32568A, 15-06049Y, PRIMUS/MED 28, UNCE204012
Ritgen:BMS: Consultancy, Other: travel support; F. Hoffmann-LaRoche: Consultancy, Honoraria, Other: travel support, Research Funding; Gilead: Other: travel support; Pfizer: Consultancy.
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