Abstract
Clinical behavior of neuroblastoma varies due to differences in biological characteristics of the tumor. 1p, 11q deletions, MYCN amplification (MNA) are known to be adverse prognostic ...markers while significance of many other copy number variations (CNV) is less clear.
We analyzed 108 primary tumors with SALSA P251 and P252 MLPA neuroblastoma kits specific for loci 1p,2p,3p,11q,17q and 100 with P253 kit (4p,7q,9p,12q,14q). Prognostic significance was estimated by event-free survival (EFS) with median of follow-up time 28 months (range 1-166 months).
MLPA revealed MNA in 19 patients (17.6%), in 12 cases (11.1%) NAG, DDX1 or ALK genes were coamplified with MYCN. EFS in case of MNA was significantly low comparing with normal MYCN status (0.20±0.11 vs. 0.69±0.06, p<0.001). 2p23-24 gain was observed in 15 patients (13.9%) and showed prognostic significance in patients under 1 year (EFS 0.53±0.25 vs. 0.96±0.04, p=0.047). In 29 patients (26.9%) 1p deletion was revealed. It had prognostic impact in the whole group (EFS 0.33±0.10 vs. 0.67±0.06, p=0.002) and in MYCN non-amplified patients (EFS 0.40±0.14 vs. 0.71±0.06, p=0.029). 17q gain was detected in 54 patients (50.0%) and led to decreased EFS in all patients (0.42±0.08 vs. 0.71±0.07, p<0.001) as well as in MYCN-negative subgroup (0.47±0.10 vs. 0.77±0.07, p=0.002). 4p gain detected in 8 patients (8.0%) dramatically decreased EFS in patients under 1 year of age (0.00 vs. 0.88±0.06, p=0.055). Gain of short and long arms of chromosome 7 detected in 6 patients (6.0%) led to reduced EFS in the whole group (0.33±0.19 vs. 0.56±0.06, p=0.053) and in MYCN non-amplified patients: 0.40±0.22 vs. 0.64±0.06, p=0.044.
In 8 patients (8.0%) 9p deletion was found. Presence of this aberration resulted in vivid decreasing of EFS in both all evaluated patients (0.00 vs. 0.60±0.06, p=0.035) and MYCN non-amplified group (0.00 vs. 0.68±0.06, p=0.047). 3p deletion observed in 30 of 108 patients (27.8%) had adverse prognostic significance in the group of patients above 3 years of age (EFS 0.25±0.23 vs. 0.71±0.11, p=0.034).
Neither interstitial (n=16, 14.8%) nor terminal (n=9, 8.3%) 11q deletions had prognostic significance in our series: EFS 0.28±0.14 and 0.78±0.14 vs. 0.60±0.06, p=0.164 and p=0.477 respectively. No influence on EFS was found in case of presence 12q or MDM2 gene gain (n= 20, 20.0%) and 14q aberrations (n=14, 14.0%).
Thus, MLPA is accurate technique for CNV detection in neuroblastoma. In our cohort of patients, treated according GPOH NB2004 protocol, MNA, 1p, 9p deletions and 17q gain demonstrated negative influence on EFS. Presence of 2p23-24 gain and 4p gain led to decrease EFS in the group of patients below 1 year old, while 1p and 9p deletions, 17q gain and gain of both arms of chromosome 7 retained prognostic significance in MYCN non-amplified patients. Coamplification of MYCN gene with DDX1, NAG and ALK genes had no prognostic significance comparing with amplification of MYCN gene alone.
Citation Format: Alexander E. Druy, Grigory A. Tsaur, Egor V. Shorikov, Alexander M. Popov, Leonid I. Saveliev, Larisa G. Fechina. Prognostic significance of genetic aberrations in neuroblastoma. abstract. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1900. doi:10.1158/1538-7445.AM2014-1900
Acute lymphoblastic leukemia (ALL) in infants (less than 1 year old) is a unique tumor with distinct biological features and poor outcome even when modern treatment schemes are used. Rearrangementsof ...MLL-gene, located in 11q23 region, are detected in approximately 75-80% of infants with ALL and lead to treatment resistance and high relapse rate. Nevertheless patients with germline MLL also demonstrate inferior outcome compared to ALL in older children. Thus additional risk factors implementation is one of the crucial points in infant ALL management. Minimal residual disease (MRD) measurement by multicolor flow cytometry (FCM) or various PCR technics is a well-standardized method of treatment response evaluation in childhood ALL, although in infants MRD data is not so widely applied. The aim of the study was to evaluate the relapse prediction feasibility in infant ALL by FCM MRD assessment during remission induction of MLL-Baby protocol.
Methods. Totally 89 infants aged from 5 days to 11 months were enrolled in the present study. In 23 cases (25.8%) MLL gene was germline (MLL-g group), 33 patients (37.1%) had MLL-AF4 fusion while in remaining 33 cases (37.1%) other types of MLL-rearrangements were found. All patients were diagnosed as B-cell precursor ALL and all were treated by well established in Russia and Belarus MLL-Baby protocol, which is specially designed for infant ALL management. MRD was measured by 6-10-color FCM in bone marrow (BM) samples obtained at day 15 and at the end of remission induction (day 36). The availability of samples at at least one of these time-points was the only criteria for study group completion. In 43 patients with known types of MLL-rearrangements MRD was also assessed by fusion gene transcript (FGT) detection in RQ-PCR after first consolidation or first high risk block (for intermediate risk and high risk groups respectively). Relapse risk was investigated by cumulative incidence of relapse (CIR) estimation. Median of follow-up was 3 years 10 months.
Results. Finally at day 15 MRD was studied in 71 cases. 17 patients (23.9%) were tested MRD-negative while remaining ones displayed various levels of MRD-positivity: 8 cases (11.3%) - from 0.01% to 0.1%; 14 (19.7%) - from 0.1% to 1%; 22 (31.0%) - from 1% to 10%; 10 (14.1%) - more than 10%. Proportion of MRD-positivity was lower in MLL-g group compared to MLL-rearranged (MLL-r) patients (58.8% and 81.5% respectively, p=0.06). Prognostic impact of day 15 MRD differed due to MLL-status. In MLL-r group significant differences between MRD(+) and MRD(-) patients were observed (n=10, CIR 0.28(0.18) and n=37, CIR 0.67(0.08) respectively, p=0.025). At the same time in MLL-g group these outcome differences were not significant (n=7, CIR 0 and n=9, CIR 0.22(0.14) correspondingly, p=0.197). Interestingly, in patients carrying MLL-AF4 fusion, known to be one of the most adverse types of MLL-rearrangements, day 15 MRD-negativity predicted low relapse incidence (n=5, CIR 0 and n=16, CIR 0.68(0.12) respectively, p=0.045). Thus day 15 MRD-negativity allows to detect low-risk MLL-r infants but it is not applicable in MLL-g group. It was previously shown that any detectable level of FGT after first consolidation or first high risk block predicts very poor outcome in MLL-r cases (G. Tsaur et al, ASH-2011). In current series RQ-PCR data in patients, FCM MRD(+) at day 15, distinguished groups of intermediate and very high relapse risk (n=17, CIR 0.51(0.13) and n=18, CIR 0.84(0.09) respectively, p=0.017). At day 36 FCM MRD was assessed in 82 infants. Among them 35 (42.7%) were tested negative while remaining 47 (57.3%) were MRD(+) at various levels. Prognostic value of day 36 FCM MRD data in MLL-r group was not significant: MRD(+) patients (n=23) had CIR of 0.71(0.08) while in MRD(-) cases (n=33) CIR was 0.49(0.12), p=0.153. Conversely, in MLL-g group low end-induction MRD (less than 0.1%) lead to excellent outcome compared to patients with higher MRD (n=14, CIR 0 and n=7, CIR 0.22(0.20) respectively, p=0.010).
Conclusions. Thus FCM MRD data could distinguish infants with low risk of ALL relapse, but in MLL-r and MLL-g groups different time-points are prognosticaly significant. In MLL-g patients tandem application of FCM at early time-point and RQ-PCR later could help to define groups with low, intermediate and high relapse risk. MRD data could be added to MLL-Baby protocol risk group stratification, which is currently based on type of MLL-rearrangement.
No relevant conflicts of interest to declare.
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10055
Background: Bone marrow (BM) involvement and MRD detection in neuroblastoma (NB) seems to be useful tool for patients’ prognosis, stratification and risk-adapted treatment. ...Real-time PCR (RQ-PCR) of tumor-specific gene transcripts and flow cytometry (FC) are commonly applied for this purpose. Aim. RQ-PCR MRD marker definition, its qualitative concordance with FC and prognostic impact in NB patients. Methods: We analyzed 331 BM samples from 57 NB patients and 26 ‘normal’ BM samples from 20 patients without malignancies for PHOX2B, TH, ELAVL4 and GD2 genes expression. BM samples were defined as positive either in case of positivity for PHOX2B or in case of NB cells presence in BM. 326 BM samples from 52 NB patients were analyzed by RQ-PCR and FC together. Results: PHOX2B and TH were not detecting in normal BM samples. Out of 224 negative BM samples TH was identified in 5 only, while ELAVL4 and GD2 expression was detected in the majority of normal and negative BM samples: 20 and 15 from 26 normal; 209 and 197 out of 224 negative samples correspondingly. TH, ELAVL4 and GD2 expression in positive BM samples was significantly higher comparing to negative and normal BM samples.Threshold levels of each gene expression were established by ROC-analysis and subsequently applied for overall correct prediction (OCP) calculation. OCP for PHOX2B and TH achieved 0.994 and 0.952, while OCP for ELAVL4 and GD2 were significantly lower: 0.828 and 0.767 respectively. Analytical sensitivity of RQ-PCR for PHOX2B achieved 1E-06, while sensitivity of FC ranged from 1E-03 to 1E-05. In 193(59.2%) out of 326 evaluated samples BM was negative by both methods. 38(11.7%) samples were negative by FC but positive by RQ-PCR for PHOX2B expression. 31(9.5%) samples were positive by FC but negative by RQ-PCR. 64(19.6%) samples were positive by both techniques. Thus qualitative concordance between RQ-PCR and FC achieved 78.8%. Conclusions: Patients with detectable PHOX2B expression have lower event-free survival comparing to patients with PHOX2B negative BM: 0.30±0.10 and 0.77±0.27 respectively, p=0.002 and overall survival is 0.51±0.11 and 0.79±0.07 correspondingly, p=0.083. Median of follow up in our NB patients series is 42 months, ranged from 1 to 156.
Background. Minimal residual disease (MRD) is powerful tool for prediction of treatment outcome in leukemia patients of various age groups, including infants with acute lymphoblastic leukemia (ALL). ...In the vast majority of cases only bone marrow (BM) samples are used for MRD detection.
Objective. To estimate prognostic significance of MRD in peripheral blood (PB) and BM by qualitative detection of different MLL fusion gene transcripts in infant ALL enrolled into MLL-Baby protocol.
Methods. Fifty three infants (20 boys and 33 girls) with median age of 5.3 months (range 0.03-11.80) and defined MLL rearrangements were included in the current study. Among them there were 25 patients (47.2%) carrying MLL-AFF1 fusion gene transcripts, 10 (18.9%) MLL-MLLT3-positive cases, 9 (17.0%) MLL-MLLT1-positive cases, 5 (9.4%) MLL-MLLT10-positive cases and 4 (7.5%) MLL-EPS15-positive ones. MRD evaluation was performed by detection of MLL fusion gene transcripts in BM and PB samples using real-time PCR and nested RT-PCR with sensitivity non-less than 1E-04. MRD-negativity was defined as absence of fusion gene transcripts in both assays. Median of follow-up period in the observed group was 5.2 years. Time points (TP) for MRD assessment were as follows: day 15 of remission induction (TP1), at the end of remission induction (TP2), after each course of ATRA administration (TP3-TP7). Informed consent was obtained in all cases.
Results. We estimated 142 paired BM/PB samples. 77 samples were double positive, 43 were double negative Thus concordance between MRD results in BM and PB samples achieved 84.5%. Concordance varied between different TPs of MLL-Baby protocol from 79.0% to 100%. The highest concordance rate was at TP4 and TP7 (92.3% and 100%, respectively). Interestingly, all discrepant results (22 samples 15.5%) were BM-positive/PB-negative. Median level of ABL gene, used for normalization, was similar in BM and PB samples (4.85E+04 vs 4.95E+04, respectively, p=0.760). Evaluation of prognostic significance of MRD in BM in TP1-TP7 revealed that TP4 was the earliest TP when discriminative data between MRD-positive and MRD-negative patients were obtained. MRD-positivity at TP4 in BM led to unfavorable outcome. Event-free survival was significantly lower in MRD-positive group (n=22) in comparison to MRD-negative one (n=31) (0.06±0.06 vs 0.70±0.09 p=0.0001), while cumulative incidence of relapse in MRD-positive patients was remarkably higher (0.92±0.01 vs 0.29±0.08, p<0.0001). MRD-positivity at this TP in BM was the only significant factor in the diagnostic model where initial risk factors (age at diagnosis, initial WBC count, immunophenotype, CNS disease, presence of MLL-AF4) were combined to response criteria (number of blast cells at day 8 of dexamethasone prophase and MRD in BM at TP4) (Table). The only TP when MRD data obtained from PB samples had prognostic value was TP6. In this TP cumulative incidence of relapse in MRD-positive patients was significantly higher in comparison to MRD-negative ones (0.88±0.11 vs 0.25±0.13, respectively, p=0.003). However these data did not bring any extra advantages as compared to TP4 in BM.
Conclusions. Despite high qualitative concordance rate between MRD detection in BM and PB samples we could not show prognostic value of MRD monitoring in PB by fusion gene transcripts. Univariate and multivariate analysis revealed that MRD-positivity at TP4 in BM was the only significant and independent prognostic factor of unfavorable outcome in the observed group of patients.
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No relevant conflicts of interest to declare.
Abstract 2547
Acute lymphoblastic leukemia (ALL) in children less than 1 year old is the relatively rare disease with specific biological features and poor outcome. It is also characterized by high ...incidence of MLL gene rearrangements. Immunophenotype of infants' leukemia varies due to presence or absence of MLL-rearrangements.
description of immunophenotype in infant acute lymphoblastic leukemia.
Totally 421 cases of pediatric acute leukemia (AL) were studied. 81 patients (39 boys and 42 girls) aged from 5 days to 11 months were included in the study group. Their data was compared to 332 cases of acute leukemia in older children. Tumor cells immunophenotyping was performed by 6–8-color flow cytometry. Detection of various types of MLL-gene rearrangements was done by simultaneous application of chromosomal banding analysis, fluorescence in-situ hybridization, reverse-transcriptase polymerase chain reaction (PCR) and long-distance inverse PCR.
There were 54 (66.7%) ALL cases in the study group. ALL was found less frequently in infants than in older children (66.7% and 88.5% respectively, p<0.0001) while percentage of acute myeloid leukemia cases was higher in infants (27.2% and 10.0% respectively, p=0.0001). EGIL immunophenotypes distribution also differed between infants and older children. BI-ALL was the most common immunological ALL type in infant ALL (55.6% vs 3.3% in older age group, p<0.0001), while BII-ALL was notably less frequent compared with other age groups (33.3% and 77.1% respectively, p<0.0001). T-lineage ALL was also less frequent in infants (3.7% vs 14.3% in older age group) although difference did not achieve statistical significance (p=0.0536). Totally infant ALL were mainly presented by B-cell precursor ALL (BCP-ALL) – 51 patients (94.4%). Various types of MLL-rearrangements were found in 40 (74.1%) patients (pts) out of 54 infants ALL cases. Among them 21 pts (52.5%) carried MLL-AF4 fusion gene, 8 pts (20.0%) – MLL-MLLT1, 5 pts (12.5%) – MLL-MLLT3, 3 pts (7.5%) – MLL-EPS15, 1 pt (2.5%) – MLL-MLLT10, 1 pt (2.5%) – MLL-AFF3 and 1 pt (2.5%) had MLL-rearrangement with unidentified partner gene. Significant immunophenotypic differences were observed in patients with and without MLL gene rearrangements. Number of cases in those tumor cells expressed CD10, CD20, CD45, CD133, CD15, NG2 significantly varied between MLL-positive and MLL-negative groups (p=0.0001, p<0.0001, p=0.0008, p=0.0018, p=0.0306 and p<0.0001 correspondingly). NG2-positivity represented the highest overall correct prediction (OCP) rate for presence of MLL-rearrangements (95.5%). Diagnostic accuracy of CD20-negativity and CD45-positivity was slightly lower (87.5% and 86.3% respectively) while OCP for CD10-negativity (78.4%), CD133-positivity (75.0%) and CD15-positivity (66.7%) was not sufficient enough. Nevertheless CD10-positive BCP-ALL with MLL-rearrangements differed from CD10(+) cases in MLL-germline group. CD10 homogeneous expression was noted in 10 out of 11 MLL-germline patients and in 1 of 10 MLL-rearranged cases (p=0.0011). Although there were found no significant differences in CD22-positive patients’ number, CD22(+)-cells percentage was significantly lower in MLL-positive cases (median 89.9%, range 25.2–99.7% and median 99.9%, range 96.0–99.9% respectively, p=0.0026). Thus CD20-negativity, CD10-negativity/low expression, high CD45, CD15, CD65 and NG2 expression, decreased CD22-expression are immunophenotypic signatures of MLL-rearranged infant ALL, although NG2 has the highest diagnostic efficacy. Interestingly there were no markers able to distinguish MLL-AF4-positive cases from patients carrying other types of MLL-rearrangements. Even NG2 expression intensity did not differ between these groups (p=0.2720). Except BCP-ALL pts, two cases of T-lineage ALL and one mature B-ALL (without other Burkitt lymphoma features) were found.
Thus immunophenotype of ALL in children less than 1 year old differs significantly from patients of older age groups. Infants' B-cell precursor ALL immunophenotype varies greatly due to the presence of MLL gene rearrangements. Complex diagnostic immunophenotyping of infants' ALL allows predicting presence of MLL rearrangements and NG2 is the most applicable single marker.
No relevant conflicts of interest to declare.
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Background. About 70% of chronic myeloid leukemia (CML) patients achieve early molecular response (BCR-ABLIS2 10% at 3-months) that lead to 5-years overall survival close to 95%. However, CML ...patients remain heterogeneous group and several studies in recent years were aimed to personalize treatment based on individual patients' characteristics. Our group previously put forward a hypothesis about the prognostic value of individual BCR-ABL declinerate in the first three months of CML therapy1,2. The ratio BCR-ABL at 3 months to baseline had chosen as 0.1 as best cut-off value to predict MMR at 12 months. The aims of this study were to validate our prognostic method in larger group of patients and compare these results according to CML prognostic scores.
Patients and methods. Fifty-five patients (median age, 52 years; range 19-84; 24 male and 31 female) with chronic phase CML were included in the study. Patients' distribution for Sokal risk groups were as follows: low-30 / intermediate-15 / high-10. Six patients had EUTOS high-risk. Forty-two patients started treatment with Imatinib 400 mg/day, 12 patients started with Nilotinib 600 mg/day and 1 patient started with Dasatinib 100 mg/day. Median BCR-ABL transcript levels was 41.38% at diagnosis, range 3.39-3185.36% (IS). The ratio of BCR-ABL levels at 3 months to baseline for each patient was calculated. In addition, we calculated ratio of BCR-ABL levels at 3 months to BCR-ABL levels at 1 month for 13 patients. Comparison was made of the predictive sensitivity to achieve early molecular response at 3 months (10% by IS) and according to prognostic CML scores (Sokal and EUTOS). We also assessed positive likelihood ratio (LR) value for the probability of achieving MMR between patients' stratification methods. Statistical analysis was conducted with Fisher exact test and sensitivity-specificity analyses.
Results. Twenty-six out of 34 patients (76.5%) with ratio of BCR-ABL levels at 3 months to baseline below than 0.1 achieved MMR at 12 months, while only 9 of 21 patients (42.9%) with ratio more than 0.1 had optimal response (LR = 1.86 (1.05 - 3.29); p=0.003). Ratio of BCR-ABL levels at 3 months to 1 month showed much better results with the same (0.1) cut-off value - 5 out of 6 patients (83.3%) with ratio BCR-ABL at 3 months to 1 month below than 0.1, while only 1 patient (14.3%) with ratio more than 0.1 achieved optimal response (LR = 5.83 (0.92 - 37.08); p=0.05), respectively. Application of early molecular response at 3 months (10% by IS) yielded worse discrimination results: 34 of 47 (72.3%) patients with BCR-ABL level ²10% at 3 months, whereas 2 of 8 (25%) patients with BCR-ABL >10% had MMR at 1 year (LR = 1.38 (1.01 - 1.89); p=0.78), respectively. CML prognostic scores results had the following sensitivity-specificity results: for Sokal - low-risk 23 of 30 (76.7%), intermediate-risk 9 of 15 (60%) and 3 of 10 (30%) high-risk patients achieved MMR at 1 year (LR (low+intermediate)/high = 1.41 (1.00 - 1.97); p=0.03); for EUTOS-score - low-risk 34 of 49 (69.4%) and only 1 of 6 (16.7%) high-risk patients had achieved MMR at 12 months (LR = 1.30 (1.00 - 1.68); p=0.02). Furthermore, application of our ratio cut-off value among patients with BCR-ABL level ²10% at 3 months allowed us to revealed additional 6 high-risk patients have not reached MMR at 1 year of therapy (Table 1).
Conclusion. Our study showed that individual rates of BCR-ABL decline from baseline to 3 months and to 1 month had better LR than CML prognostic scores (Sokal, EUTOS) or early molecular response achievement (BCR-ABL levels ²10% at 3 months) and might be useful as an optimized predictors of outcome for CML patients (MMR at 1 year of treatment).
1 Fominykh M., ShuvaevV., Martynkevich I. et al. ELN Frontiers Meeting ÇWhere science meets clinical practiceÈ 16-19 October, 2014, Berlin, Germany. Abstract book: 11.
2 Shuvaev V., Fominykh M., Martynkevich I. et al. Blood (56th ASH Annual Meeting Abstracts), 2014; 124 (21): 5529.
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Chelysheva:Novartis Pharma: Consultancy, Honoraria; Bristol Myers Squibb: Honoraria. Turkina:Bristol Myers Squibb: Consultancy; Pfizer: Consultancy; Novartis Pharma: Consultancy.
Acute lymphoblastic leukemia (ALL) in infants is a relatively rare disease with peculiar biological features and worse outcome in comparison to ALL in older children. Infant ALL is characterized by a ...high frequency of MLL gene rearrangements, mainly CD10-negative B-cell precursor ALL (BCP-ALL) immunophenotype and high tumor burden at diagnosis. Even with new therapeutic approaches event-free survival (EFS) in this subgroup of patients does not exceed 50%. Although flow cytometric (FCM) minimal residual disease (MRD) detection at day 15 of remission induction is well established for patients' stratification in older children treated with the AIEOP-BFM-2009 protocol, the prognostic value of FCM MRD in infant ALL is not fully known yet.
Aim of the present study was to evaluate the prognostic significance of FCM MRD measurement in infants with ALL treated with Interfant-99 and Interfant-06 protocols in AIEOP (Associazione Italiana Ematologia Oncologia Pediatrica) centers in Italy.
Between May 1999 and December 2011, 120 consecutive infants aged 0 to 365 days with newly diagnosed ALL were treated in AIEOP centers with the Interfant99 and the on-going Interfant-06 protocols. Among these patients, 51 (42.5%) with available day 15 follow-up bone marrow samples were included in this study on FCM MRD. In 39 (76.5%) cases, different types of MLL gene rearrangements were identified by fluorescence in situ hybridization (FISH), while 12 (23.5%) patients had germline MLL. MRD detection was performed by 4-6-color FCM. Median follow-up time was 3.5 years (range: 1 month – 7.5 years). Outcome was estimated by evaluating the probability of EFS and the cumulative incidence of relapse (CIR). Analysis of prognostic relevance of FCM MRD in combination with other criteria used for stratifying patients enrolled in the Interfant-06 protocol was performed with the Cox model on the cause-specific hazard of relapse.
We classified infants according to the AIEOP-BFM day 15 stratification into three risk groups: 14 patients (27.5%) were considered at standard risk (SR: MRD less than 0.1%), 9 patients (15.7%) at high risk (HR: MRD 10% or more), and the majority of infants (29, 56.9%) at intermediate risk (IR: MRD 0.1% to 10%). As the 14 SR patients had 3-year EFS and CIR significantly better than other patients, we considered two major groups of patients with different outcome: SR group (MRD<0.1%) with 3-year EFS 77.9% (standard error, SE, 11.3) and CIR 14.9% (SE 10.2), and non-SR group with 3-year EFS 32.0% (SE 8.5) and CIR 58.0% (SE 8.8, p=0.0104 and p=0.0085, respectively). Half of SR group (7 of 14 cases) had germline MLL. 4 out of 7 MLL-positive SR-patients were in continuous complete remission (CCR) In contrast, the majority of infants in the non-SR group carried various types of MLL rearrangements. Only 5 cases in the non-SR group were MLL germline and only two of them are still in CCR. We evaluated the prognostic impact of day 15 MRD in MLL-positive cases (n=39). In this cohort of patients, we also observed a difference, although not statistically significant, between SR and non-SR groups both in 3-year EFS (57.1%, SE 18.7 and 30.9%, SE 9.2, respectively; p=0.3630) and in 3-year CIR (28.6%, SE 18.9 and 60.9%, SE 9.5, respectively; p=0.1733). We evaluated the suitability of MLL negativity and of day 15 FCM MRD <0.1% as single criterion for the identification of low-risk patients. Each factor, when separately analyzed in a Cox model, was significantly correlated with a reduction in the risk of relapse, as shown in Table 1, left panel. Nevertheless, as day 15 FCM MRD levels are strictly related to MLL status, the Cox model which analyzes jointly the two factors, is unable to identify the one independently impacting on the risk of relapse (Table 1, right panel). Thus, although being a strong prognostic factor by itself, day 15 FCM MRD stratification did not confer an advantage in relapse prediction when considered in combination with MLL status, which is the only low-risk group criterion in the Interfant-06 stratification.
Day 15 FCM MRD proved to be a suitable variable predicting treatment failure and can be used as an alternative or in combination with Interfant-06 stratification criteria to identify SR patients.
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Popov:Alexion: Research Funding.
The KMT2A gene (previously known as MLL ) located at 11q23 is often involved in recurrent chromosomal translocations that lead to the development of acute leukemia, particularly in infants. Acute ...leukemias with KMT2A rearrangements have different prognoses, which depend on the partner gene involved in the translocation. The detection of all possible types of KMT2A gene rearrangements is of key importance for the identification of biological subgroups, which may differ in clinical outcome. In this report, we describe a case study of a 7-month-old boy who presented with AML-M4; however, no obvious 11q23 rearrangement was detected in the analyzed karyotype. Fluorescence in situ hybridization evaluation showed a nonstandard signal distribution in blast cells, corresponding to the presence of two KMT2A copies and one additional copy of 5′-KMT2A inserted into the long arm of the X chromosome (ins(X;11)(q28;q23q23)). Subsequent molecular analysis showed a novel variant form of the previously described KMT2A-FLNA fusion gene, in which the KMT2A intron 9 is fused to the FLNA exon 16.
Abstract 1449
Central nervous system (CNS) involvement is one of the important risk factors in childhood acute leukemia (AL). Tumor cells detection in cerebrospinal fluid (CSF) is one of the main ...signs of CNS lesion. Traditionally blasts presence in CSF is assessed by conventional cytomorphology (CM) of cytospin slides. However, sensitivity of this method is relatively low. Flow cytometry (FC) having a higher sensitivity could provide better diagnostic applicability for CSF blasts detection. Aim. To compare results of tumor cells detection in CSF of children with AL by flow FC and CM. Methods. 183 samples from 52 boys and 31 girls aged from 5 months to 15 years with different types of acute lymphoblastic leukemia (ALL) (77 patients), acute myeloid leukemia (AML) (5 patients) and acute biphenotypic leukemia (1 patient) were investigated. 17 positive samples obtained by traumatic lumbar puncture were excluded from analysis because tumor blasts were also detected in peripheral blood. Comparison between FC and CM data was performed in 166 samples. Among these samples 61 was taken at the time of initial diagnostics, 34 – during AL follow-up, 17 – at relapse and 54 – during relapse monitoring. Monoclonal antibodies panels were constructed according to immunophenotype of tumor cells in bone marrow. Results. In 24 out of 166 samples (14.5%) tumor cells were detected by CM. In all these cases blasts were also found by FC, while FC allowed finding blasts in other 35 samples. Thus the total number of FC-positive samples was 59 out of 166 (35.5%). This frequency was significantly higher than rate of CM-positive cases (g < 0.0001). Among initial diagnostics samples there were 20 FC-positive and only 10 CM-positive patients (32.8% vs. 16.1%, p=0.0585). At relapse 9 (52.9%) patients were FC-positive, while 6 (35.3%) were CM-positive (p=0.4897). In both B-lineage and T-lineage ALL, analyzed separately, FC detected blasts in CSF frequently than CM (p=0.0098 and p=0.0002 respectively). Absolute blast count in 1 ml in CSF samples, positive by both methods was significantly higher than in samples, positive only by FC (median = 418, range 8–158171 and median = 34, range 5–2762 respectively, g = 0.0002). Thus FC allows detecting tumor cells in CSF much more frequently than conventional CM, which could be explained mainly by higher FC sensitivity. Moreover FC is applicable also for qualitative and quantitative monitoring of CNS lesion. Nevertheless prognostic impact of FC CSF investigation is questionable. Among 13 patients in whom discordant results were obtained in initial diagnostics samples and at relapse, only for one patient risk stratification could have been changed. For all other patients there were other risk factors, that decreased significance of FC leukemic blast detection in CSF. Conclusion. Flow cytometry allows more frequent detection of tumor blasts in CSF of children with AL, while prognostic significance of these findings is still unclear and needs to be confirmed in large prospective trials.
No relevant conflicts of interest to declare.
Infant acute leukemia is characterized by high incidence of MLL gene rearrangements.
To evaluate the distribution of MLL genomic DNA breakpoints and their relation to several diagnostic parameters ...among infant acute leukemia.
72 infants with MLL-rearranged acute lymphoblastic leukemia (ALL) (n=52), acute myeloid leukemia (AML) (n=19) and mixed phenotype acute leukemia (n=1) were included in this study based on the availability of DNA material at diagnosis. In the observed group there were 28 boys (39%) and 44 girls (61%) with median age of 4.9 mo (range 0.03-11.9). Genomic DNA breakpoint detection in MLL gene and translocation partner genes (TPG) was performed by long-distance inverse PCR (LDI-PCR). Exon-intron numbering of MLL gene was done according to I. Nilson et al, 1996.
Majority of ALL cases (n=28; 54%) was characterized by presence of MLL-AF4 fusion gene (FG), less frequently MLL-MLLT1 (n=12; 23%), MLL-MLLT3 (n=7; 13%) and others were found (Table 1). The most common breakpoint location within MLL gene in ALL patients was intron 11, detected in 25 cases (48%). The highest variability of MLL breakpoints was found in MLL-AF4-positive patients: only 11 of 28 (39%) had breakpoints in intron 11. The most stable pattern of MLL genomic DNA breakpoints was observed in MLL-MLLT1-positive patients: 8 of 12 (67%) had breakpoints in intron 11. In AML patients two the most prevalent FGs were MLL-MLLT3 (n=7, 37%) and MLL-MLLT10 (n=5, 26%). The remaining ones are listed in Table 1. The most frequent breakpoints location was intron 8 (8 out of 19, 42%). The most stable pattern was revealed for MLL-MLLT10 FG: MLL breakpoints in 4 of 5 (80%) cases were found in intron 9 (Table 1). ALL patients who had breakpoints in intron 11 were significantly younger (median 3.0 mo, range 0.03-11.6) than all others (median 5.6 mo, range 0.7-11.9) (p=0.025) and than patients with MLL breakpoints in intron 9 (median 6.6 mo, range 3.1-11.9) (p=0.017). For AML cases we did not find any relation between age and breakpoints locations. Distribution of MLL DNA breakpoints was similar in boys and girls and did not depend on type of TPG. Genetic recombinations involving MLL gene predominantly resulted in reciprocal chromosomal translocations (n=62; 86%). Beside them, 6 (11%) insertions were identified in all MLL-MLLT10-positive cases and MLL-SEPT6-positive one. In 11 (15%) patients we found breakpoints within the regions located from 0.7 Kb to 25.4 Kb 3' of the first exon of TPGs (MLLT1 n=9; EPS15 n=1; MYO1F n=1), however fusion transcripts at cDNA level were identified and sequenced in all these cases, indicating a spliced fusion mechanism. 3-way translocations were found in 5 patients and in 1 case we found combination of insertion with interstitial deletion of chromosome 11. The list of reciprocal genes involved in these 6 cases was as follows: CEP164, DNAH6, DCPA1, MCL1 as well as non-coding regions of 2q21.2 and 2p21. We also analyzed breakpoints in TPGs. Except above mentioned spliced fusion cases, the remaining 3 breakpoints in MLLT1 as well as 3 of 4 breakpoints in EPS15 and all breakpoints in MLLT11 were within intron 1 of corresponding genes. In AF4 the major breakpoint region included intron 3 (n=19), intron 4 (n=6) and intron 5 (n=2). We also revealed 2 rare breakpoints in intron 6 and 10. In MLLT3 the most frequent breakpoint location was intron 5 (n=12), additionally 2 cases in intron 5 were identified. In MLLT10 two separate breakpoint locations were found: intron 3 (n=1) and intron 8 (n=3) in combination with intron 9 (n=1). We estimated prognostic significance of MLL breakpoint locations in 31 cases of infant ALL treated by MLL-Baby protocol. 3-year cumulative incidence of relapse was remarkably higher in patients with breakpoints in intron 11 (n=18) in comparison to patients with breakpoint localized from intron 7 to exon 11, inclusively (n=13) (0.85±0.01 and 0.57±0.02, respectively), although difference between these two groups did not achieve statistical significance (p=0.261). Median follow-up time in the observed group was 30 months (range 6–42).
In the current study we estimated clinical and prognostic significance of MLL and TPG genomic DNA breakpoints in infant acute leukemia. Our data provide additional information of molecular genetic features of MLL-rearranged infant acute leukemia.
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No relevant conflicts of interest to declare.
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