Abstract only
e22014
Background: Rearrangements of TERT gene with its correspondent hyperexpression were identified as adverse prognostic markers in neuroblastoma (NB) patients, while significance of ...microRNAs expression is undiscovered. Methods: RNA from 103 fresh-frozen NB tissues was subjected to reverse transcription qPCR for miR128A and TERT expression evaluation. Copy number variations were determined by MLPA and FISH. Correspondence of miR128A and TERT expression levels to event-free survival (EFS) was proved by ROC-analysis and established threshold levels (TL) were utilized for group separation in subsequent survival analysis. Median of follow-up time achieved 5.8 years. Results: Abundant TERT expression and lack of miR128A expression resulted in superior frequency of adverse events (p = 0.027, TL = 4,7E-3 and p = 0.004, TL = 4.6E-2 respectively). EFS in group of patients with TERT expression above 4,7E-3 (group TERT) was 0.66SE0.07, in patients with miR128A expression below 4.6E-2 (group miR128A) was 0.64SE0.15, patients harboring both TERT overexpression and lack of miR128A expression (group miR128/TERT) had dismal outcome: EFS 0.29SE0.11, comparing to patients without these abnormalities (group "neither"): EFS 0.92SE0.06, p < 0.001. Analogously, cumulative incidence of progression differed significantly between all these groups, p < 0.001. MYCN amplified (MNA) samples were accumulated in the groups TERT and miR128/TERT (p = 0.061), while chromosome 17 gain, 9p and 14q deletions prevailed in the group "neither" (p = 0.014, 0.043 and 0.017 accordingly). MNA and 14q deletion had prognostic significance in the correspondent groups (p < 0.001, p = 0.022). The classifier was proposed for distinguishing patients with unequal outcome: MNA EFS = 0.25SE0.11, MYCN single copy patients divided into groups miR128A/TERT (0.40SE0.15), miR128A (0.60SE0.15) and TERT (0.74SE0.08). Group "neither" was separated basing on presence (0.71SE0.17) and absence (EFS = 1.00) of 14q deletion, p < 0.001. Conclusions: Levels of miR128A and TERT expression together with cytogenetic data allow discriminating patients into groups with significantly different outcome.
Sequential monitoring of minimal residual disease (MRD) by molecular techniques or multicolor flow cytometry (MFC) has emerged over the past two decades as the primary tool to optimize treatment in ...pediatric B‐cell precursor acute lymphoblastic leukemia (BCP‐ALL). The aim of our study was to compare the prognostic power of repeated MFC–MRD measurement with single‐point MRD assessment in children with BCP‐ALL treated with the reduced‐intensity protocol ALL‐MB 2008. Data from consecutive MFC–MRD at day 15 and day 36 (end of induction, EOI) were available for 507 children with Philadelphia‐negative BCP‐ALL. They were stratified into standard risk (SR, n = 265), intermediate risk (ImR, n = 211), and high risk (HR, n = 31) according to the initial clinical characteristics defined in the ALL‐MB 2008 protocol. Quantitative (relative to quantitative thresholds) and kinetic (logarithmic reduction) assessments of MFC–MRD at both time points effectively separated patients into three groups with different risk of recurrence. On the other hand, starting with low (for the SR group) and moderate (for the ImR group) induction therapy, a single MFC–MRD measurement at EOI proved sufficient to unequivocally identify patients in whom this therapy is highly effective and distinguish them from those who cannot be successfully treated with such therapy. Therefore, initiating treatment with low or moderate treatment from the start, together with careful consideration of initial clinical risk factors and just one EOI–MFC–MRD measurement is simple, inexpensive, and entirely sufficient for treatment optimization. Furthermore, for a large proportion of patients, this approach allows better adjustment, in particular also reduction of therapy intensity than sequential MRD measurements.
The aim of this study was to present the diagnostic and outcome characteristics of infants with germline status of KMT2A gene (KMT2A‐g) B‐cell precursor acute lymphoblastic leukemia (BCP‐ALL) treated ...consistently according to the MLL‐Baby protocol, a moderate‐intensity protocol. Of the 139 patients enrolled in the MLL‐Baby study, 100 (71.9%) carried different types of rearranged KMT2A (KMT2A‐r), while the remaining 39 infants (28.1%) had KMT2A‐g. KMT2A‐g patients were generally older (77% older than 6 months), less likely to have a very high white blood cell count (greater than 100 × 109/L), less likely to be central nervous system (CNS)‐positive, and more likely to be CD10‐positive. The 6‐year event‐free survival and overall survival rates for all 39 patients were 0.74 (standard error SE 0.07) and 0.80 (SE 0.07), respectively. Relapse was the most common adverse event (n = 5), with a cumulative incidence of relapse (CIR) of 0.13 (SE 0.06), while the incidence of a second malignancy (n = 1) and death in remission (n = 3) was 0.03 (SE 0.04) and 0.08 (SE 0.04), respectively. None of the initial parameters, including genetics and the presence of recently described fusions of NUTM1 and PAX5 genes, was able to distinguish patients with different outcomes. Only rapidity of response, measured as minimal residual disease (MRD) by flow cytometry, showed a statistically significant impact. Moderate‐intensity therapy, as used in the MLL‐Baby protocol in infants with KMT2A‐g BCP‐ALL, yields results comparable to other infant studies. Patients with a slow multicolor flow cytometry (MFC)‐MRD response should be subjected to advanced therapies, such as targeted or immunotherapies.
Purpose
Multicolor flow cytometry (MFC) is widely available, fast and has an easy-to perform approach for finding neuroblastoma (NB) cells among normal bone marrow (BM) hematopoietic cells. Aim of ...the study was to investigate prognostic significance of initial MFC tumor cells’ detection in BM of children with NB.
Methods
51 patients (24 boys and 27 girls) aged from 6 days to 15 years (median age 1 year 3 months) with NB were included in the study. BM samples at the time of diagnosis were obtained from 2 to 5 aspiration sites per patient. CD45(−)CD56(+)CD81(+)GD2(+)-cells were evaluated by MFC.
Results
NB cells were detected in BM by FC more frequently compared to conventional cytomorphology (49.0% and 29.4% patients, respectively,
р
= 0.043). Patients with NB cells detected in BM by MFC had significantly worse event-free survival and cumulative incidence of relapse/progression 0.24(0.08) and 0.60(0.10), respectively compared to children with negative result of immunophenotyping 0.85(0.07) and 0.12(0.06), respectively,
p
< 0.001 in both cases. BM involvement detection by MFC maintained its prognostic significance in various patients groups. In multivariate analysis, immunophenotyping proved to be an independent prognostic factor when analyzed jointly with other NB risk factors. In 42 patients BM involvement was also studied by RQ-PCR for
PHOX2B
and
TH
genes expression. Within groups of patients divided by RQ-PCR positivity, MFC-positivity retained prognostic significance.
Conclusions
Thus flow cytometric BM involvement detection has very strong prognostic impact even stronger than RQ-PCR. It could be used in combination with other parameters for the treatment strategy choice in patients with NB.
Conventionally central nervous system (CNS) involvement in acute lymphoblastic leukemia (ALL) is found by blasts detection in cerebrospinal fluid (CSF) via cytospin slides microscopy. It was shown ...earlier, that low levels of tumor cells, undetectable by cytomorphology (CM), could be found in several ALL cases using more sensitive techniques such as multicolor flow cytometry (MFC) (A. Popov et al, ASH-2011). Nevertheless prognostic value of these low levels of blasts in CSF is still unclear. The aim of present study was to evaluate the clinical significance of MFC application for initial diagnostics of leukemic cells in CSF of children with ALL.
Patients and methods. Since December 2008 till November 2014 in 155 consecutive children older than 1 year with ALL initial CNS involvement was investigated by CSF flow cytometry. Study group included 65 girls and 90 boys aged from 1 to 16 years (median age 4 years) treated according to ALL MB-2008 protocol. 134 patients (86.5%) suffered from various types of B-cell precursor ALL (BCP-ALL), while remaining 21 cases (23.5%) were T-lineage ALL. According to the protocol stratification system 59 patients belonged to standard risk group (SRG), 87 - to intermediate risk group (ImRG) and 9 - to high risk group. CSF lesion was studied in parallel by microscopy of cytospin slides and by MFC. CNS status was classified as CNS1 (no blast cells in CSF), CNS2 (<5 WBC/ul CSF with blast cells), or CNS3 (≥5 WBC/ul CSF with blast cells, or other signs of CNS involvement). Antibodies' combinations for cytometric CSF assessment were developed on the basis of leukemic cells immunophenotype defined by initial diagnostic bone marrow investigation. Treatment outcome was estimated by cumulative incidence of relapse (CIR). Median of follow up was 5 years.
Results. In 5 patients CNS3 status was defined. These patients were excluded from the further analysis due to obvious neuroleukemia signs. CNS2 was found in 23 of 150 remaining cases (15.3%) while 127 (84.7) had CNS1. Totally 53 out of 150 studied children (35.3%) had MFC-detectable CSF lesion. In 32 CNS1-patients leukemic blasts were found only by MFC. Absolute blast count in 1 ml in CSF samples, positive by both methods was significantly higher than in samples, positive only by MFC (median = 418, range 8-158171 and median = 34, range 5-2762 respectively, p<0.001). Thus MFC allows detecting tumor cells in CSF much more frequently (p<0.001) than conventional CM. This difference could be explained mainly by higher MFC sensitivity. Both CM and MFC presented results, valuable for outcome prediction. Children with CNS2 (n=23) had higher relapse incidence that patients with CNS1 (n=127): CIR 0.38(0.11) and 0.16(0.05) respectively, p=0.005. 53 MFC-positive patients displayed higher CIR compared to 97 MFC-negative ones: 0.26(0.06) and 0.15(0.05) respectively, p=0.019. Nevertheless MFC lacked prognostic impact in clinically relevant patients' groups. Among CNS1 cases the CIR difference between MFC-positive (n=32) and MFC-negative (n=95) groups was not significant: 0.19(0.07) and 0.15(0.05) respectively, p=0.148. SRG patients, who could be restratified according to the results of CSF inspection, also haven't displayed any difference in relapse incidence between 51 MFC-negative patients (CIR 0.18(0.07)) and 8 MFC-positive ones (CIR 0.27(0.17), p=0.313). In ImRG after exclusion of T-ALL cases cytometric CSF assessment also lacked its prognostic impact: MFC-negative group (n=33) had similar outcome to MFC-positive patients (n=33): CIR 0.08(0.06) and 0.17(0.07) respectively, p=0.239. In 37 initially MFC-positive cases follow-up CSF samples were also studied. Patients with fast clearance of CNS burden (n=24) displayed better outcome compared to children who remained MFC-positive (n=13), although these differences didn't achieve statistical significance: CIR 0.32(0.10) and 0.54(0.14) respectively, p=0.116.
Conclusions. Our data shows that MFC is able to find in CSF even small number of leukemic blasts, which are undetectable by conventional cytospin slides microscopy. Despite of this advantage of cytometric CSF inspection, the prognostic value of MFC data remains controversial. In contrast, larger tumor cells populations that could be detected in CSF also by cytomorpology are more strictly associated with high incidence of relapse. Nevertheless this data should be confirmed on larger patients' cohort in multicenter trial.
No relevant conflicts of interest to declare.
Aim:
to determine the frequency of
PNPLA3
rs738409 C>G gene polymorphism, leading to
p
.I148M substitution, in patients with non-alcoholic fatty liver disease (NAFLD), and to reveal the association ...between polymorphism and probable NAFLD outcomes: liver cirrhosis (LC) and hepatocellular carcinoma (HCC).
Materials and methods.
The study was conducted according to the “case-control” design, three main groups were formed: a group with NAFLD (
n
= 46), a group with LC (
n
= 61), a group with HCC (
n
= 50), as well as a control group (
n
= 70), for all groups we performed genotyping of the rs738409 polymorphism of the
PNPLA3
gene. The relationship between the occurrence of different genotype variants and the diagnosis of patients was evaluated, the odds ratio (OR) of progression of NAFLD and the reliability of intergroup differences were determined.
Results.
NAFLD patients with
PNPLA3
I148M polymorphism have a significantly higher chance of developing LC and HCC. The odds ratio for the GG genotype was 7.94 (95 % Cl: 2.19–28.84;
p
= 0.030) for LC and 6.51 (95 % Cl: 1.15–4.08;
p
= 0.039) — for HCC with concomitant LC. The presence of the minor G allele also increases the likelhood of transition from NAFLD to LC (OR = 2.38; 95 % Cl: 1.41–4.02;
p
= 0.010) and HCC in the presence of cirrhosis (OR = 2.17; 95 % Cl: 1.15–4.08;
p
= 0.039). Differences in the frequency of
PNPLA3
polymorphism between the NAFLD and HCC groups were not significant. Additional risk factors for HCC associated with NAFLD are overweight (OR = 5.14; 95 % Cl: 1.94–13.67;
p
< 0.001), arterial hypertension (OR = 8.49; 95 % Cl: 3.05–23,62;
p
< 0.001) and diabetes mellitus (OR = 8.57; 95 % Cl: 1.03–71.48;
p
= 0.032).
Conclusion.
The frequency of single nucleotide polymorphism
PNPLA3
significantly differs in patients with NAFLD, cirrhosis and HCC compared with the control group of healthy volunteers. The
PNPLA3
I148M polymorphism increases the incidence of NAFLD progression to cirrhosis and HCC, but only with concomitant cirrhosis.
Background. In 2016 WHO classification of myeloid neoplasms and acute leukemias a new provisional entity ‘B-lymphoblastic leukemia/lymphoma, BCR-ABL1-like’ has been introduced. Two current strategies ...of BCR-ABL1-like ALL detection are based on gene expression profile revealed either by microarray ('BCR-ABL-like') or by NGS / patented TaqMan low-density array (TLDA) ('Ph-like'). Although both techniques and not widely applicable. Aims. To find out whether expression profile based on combined expression data of 5 genes assessed by real-time PCR can be used for the identification of BCR-ABL1-like pediatric ALL patients. Methods. The study was done on initial bone marrow samples of 147 primary pediatric BCP-ALL patients. Positive cohort included 10 BCR-ABL1-positive ALL patients, negative cohort consisted of 59 cases with known structural or numerical aberrations. Among them there were 21 patients with high hyperdiploidy, 1 low hypodploid, 1 near tetraploid and 1 iAMP21 cases, 23 patients with translocation t(12;21)(p13;q22), 5 cases with t(1;19)(q23;p13), 1 case with t(9;11)(p22;q23) and 6 Down syndrome (DS-ALL) patients. The rest 78 cases were called 'B-others' ALL and designated as training cohort. According to the previously published data (R. Harvey et al. ASH 2013 abs #826.) we picked 5 genes (IGJ, SPATS2L, MUC4, CRLF2, CA6) those expressions were estimated by real-time PCR. ΔCt method was applied in relation to ABL1 expression. In case of having expression results similar to BCR-ABL1-positive cases, patients with BCR-ABL1-negative ALL were attributed as BCR-ABL1-like ALL. In 113 patients we evaluated IKZF1 status by MLPA using P335 kit (MRC-Holland). Presence of ABL1, ABL2, CRLF2, IgH, JAK2, PDGFRb/CSFR1 gene rearrangements were done by FISH. Prognostic significance of BCR-ABL-like profile was estimated in 66 ‘B-others’ patients uniformly treated according to the ALL-MB 2008 protocol. Informed consent was obtained in all cases. Results. Hierarchical cluster analysis and principal component analysis showed that 16 examined samples were clustered together with 9 BCR-ABL1-positive ones. Among them there were 3 DS patients, 1 iAMP21, 1 t(12;21) cases and 11 patients from ‘B-other’ group. IKZF1 deletions and high CRLF2 expression were more frequent in BCR-ABL1-like group in comparison to non-BCR-ABL1-like (67% vs 11%, and 40% vs 6%, correspondingly; p<0.001 in both cases). Among 9 BCR-ABL1-like cases which were assessed by FISH, JAK2 rearrangements were found in 2, CRLF2 in 4 (2 cases of CRLF2-IgH and 2 CRLF2-P2RY8). In 3 patients none of the tested gene rearrangements were revealed. We did not detect any tyrosine kinase fusion genes in 64 non-BCR-ABL-like ‘B-other’ patients (p<0.001). BCR-ABL1-like profile was associated with female gender (80% vs. 57% p=0.011), high initial WBC (≥30*10^9/L) (67% vs. 18%, p=0.001), M3 bone marrow status on day 15 of induction remission (27% vs. 4%, p=0.010). BCR-ABL1-like ALL patients more often were stratified to high-risk group (40% vs. 9%, p=0.001). EFS of BCR-ABL1-like patients was remarkably lower in comparison to non-BCR-ABL1-like ‘B-other’ patients enrolled into ALL-MB 2008 protocol (0.28±0.17 vs. 0.93±0.04, p<0.0001) while cumulative incidence of relapse was significantly higher (0.57±0.19 vs. 0.02±0.02, p<0.0001) with median of follow-up period 4.9 years. The worst outcome was noted in case of combination of BCR-ABL1-like profile and IKZF1 deletions (EFS 0.00), while patients with isolated BCR-ABL1-like profile doing much better (EFS 0.67±0.22). Poor EOI flow-MRD response (>0.1%) together with BCR-ABL1-like profile identified a group of patients with dismal outcome (EFS 0.00 vs 0.88±0.11, p<0.001). Overall accuracy of our BCR-ABL1-like profile for relapse prediction in ‘B-other’ group on ALL-MB 2008 protocol was 0.939 (95% CI 0.881-0.997), for risk of unfavorable event 0.909 (95% CI 0.840-0.978). Conclusion. Thus, we showed that described real-time PCR technology based on expression data of 5 genes allowed to detect the BCR-ABL1-like ALL patients with similar clinical characteristics, genetic parameters and treatment outcome to ones revealed by microarray, NGS or TLDA techniques. We believe that detection of BCR-ABL1-like profile by PCR can be used as fast and easy screening method in ‘B-other’ ALL for the further identification of tyrosine kinase fusion genes or other targetable lesions by NGS or FISH within this group only.
No relevant conflicts of interest to declare.
Aim
The aim of the study was to evaluate the incidence and prognostic impact of central nervous system (CNS) involvement in infants with B‐cell precursor acute lymphoblastic leukemia (BCP‐ALL), as ...well as its relation with minimal residual disease (MRD) data.
Methods
A total of 139 consecutive infants with BCP‐ALL from the MLL‐Baby trial were studied. Cerebrospinal fluid (CSF) samples were investigated by microscopy of cytospin slides. MRD was evaluated according to the protocol schedule by flow cytometry and PCR for fusion gene transcripts (FGT).
Results
Involvement of the CNS at any level was found in 50 infants (36.0%). The incidence of CNS involvement was higher in patients with KMT2A gene rearrangements (44.0% for KMT2A‐r vs. 15.4% for KMT2A‐g, p = .003). The outcome of CNS‐positive infants was significantly worse than that of CNS‐negative infants, although this prognostic impact was limited to the KMT2A‐r group (event‐free survival 0.21 for CNS‐positive vs. 0.48 for CNS‐negative infants, p = .044). CNS‐positive infants could not be treated successfully by conventional chemotherapy alone, irrespective of the rapidity of MRD response. In contrast, the combination of initial CNS negativity and FGT‐MRD negativity identified a group comprising up to one‐third of infants with KMT2A‐r ALL who can be treated with chemotherapy and achieve very good outcomes (disease‐free survival above 95%), and remaining patients should be allocated to receive other types of treatment.
Conclusion
We can conclude that this combination of initial CNS involvement and MRD data can significantly improve risk‐group allocation in future clinical trials enrolling infants with ALL.
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