Value of flow cytometric analysis of peripheral blood samples in children diagnosed with acute lymphoblastic leukemia

Peripheral blood samples are frequently screened by flow cytometry before bone marrow for suspected leukemia to facilitate treatment decisions. Criteria to establish the diagnosis of a lymphoblastic malignancy from peripheral blood are not well defined.
Retrospective comparison of paired results of peripheral blood flow cytometry and bone marrow in 383 children with ALL diagnosed consecutively at a single center from January 2007 to February 2016. Patients were aged 0-18 years and had an adequate peripheral blood sample collected up to 7 days prior to the BMA. Four-color flow cytometry was used until September 22, 2014, then a 10-color panel was established thereafter.
Of 383 patients with B or T precursor ALL and paired results, only 3 patients had discordant results. There were 2 false positives peripheral blood samples (corresponding to lymphoblastic lymphoma with BM involvement below the threshold of leukemia and ALL that initially did not meet diagnostic criteria but later progressed) and one false negative (qualitatively positive upon review). In 75% of patients (289/383) who underwent both peripheral blood flow cytometry and BMA, the diagnostic LP and first dose of IT chemo were performed during the same sedation as the BMA.
Hematopathologist experience may lead to bias.
High concordance of results between peripheral blood and bone marrow flow cytometry in diagnosis of ALL. 1% or more blasts in the peripheral blood anticipated a diagnosis of ALL in the subsequent BMA with high sensitivity and specificity. Peripheral blood should not replace bone marrow in the diagnosis of ALL but integration into diagnostic approach could help with scheduling initial procedures, reduce anesthesia procedures, and optimize use of healthcare resources.

Brentuximab vedotin plus bendamustine in relapsed or refractory Hodgkin’s lymphoma: an international, multicentre, single-arm, phase 1–2 trial

The objective of this study was to explore the safety and clinical activity of brentuximab vedotin plus bendamustine in heavily pretreated patients with relapsed or refractory Hodgkin's lymphoma and anaplastic large T-cell lymphoma.
This was an international, multicentre, single-arm, phase 1-2 trial. Patients with Hodgkin's lymphoma or anaplastic large-T-cell lymphoma who had received at least one previous multi-agent chemotherapy regimen.
The trial occurred in 2 phases:
Phase 1: patients were assigned following a 3+3 dose-escalation design to one of four cohorts to receive brentuximab on day 1 of a 21-day cycle and bendamustine on days 1 and 2 of a 21-day cycle. Outcomes were maximum tolerated dose and dose limiting toxicity.
Phase 2: all patients received brentuximab plus bendamustine at the recommended dose from phase 1.
Complete response was defined as PET negativity.
65 patients were enrolled, most with Hodgkin's lymphoma (n=64). The age range of patients was 18-72 years. 28 patients were evaluated in the phase 1 study and 37 patients were evaluated in the phase 2 study. Maximum tolerated doses were not reached. Dose limiting toxicities included grade 4 neutropenia and diffuse rash. The dose used for phase 2 was the standard dose of each agent when used independently. An overall response was achieved in 29/37 (78%) of patients. Complete response was obtained in 16/37 patients (43%).
6 patients in the phase 1 study and 3 patients in the phase 2 study had progression of disease during treatment (9/65, 14%).
This was a non-randomized trial in a heavily pre-treated group of patients. There are only few patients included in this study. Only adults participated in this study with a range of ages from 18-72 years.
Brentuximab vedotin and bendamustine achieved a clinical response in relapsed and refractory adult patients with Hodgkin's disease.

Young Female Donors Do Not Increase the Risk of Graft-versus-Host Disease or Impact Overall Outcomes in Pediatric HLA-Matched Sibling Hematopoietic Stem Cell Transplantation.

This is a retrospective cohort study, in 244 pediatric patients, to address the hypothesis that the presence of T and B cells sensitized by exposures during pregnancy are a contributing factor to differences in outcomes between sex-matched and sex-mismatched transplants. Theoretically, there should be no such difference when a non-exposed (non-alloimmunized) female donor is used. In the study, they assume that young (12 years) female donors are a sexually naïve population and therefore the presence of alloimmunization including to H-Y antigens should be minimal.
Data from 244 pediatrics patients were analyzed. The outcome was the development of acute grade II to IV GVHD and Chronic GVHD. Survival analysis was assessed at 100 days, 1 year and 5 years. Age was dichotomized to improve the interpretability of the results.
Donor age >12 yrs represents 50% of the population in the study. Univariate analysis revealed older patient age, older donor age, conditioning with CY-TBI and earlier year of transplant as significant predictor of aGVHD. Of these, all but donor age showed significance in multivariate analysis.
The effect of female donor sex on cGVHD noted in the model adjusted for patient age, HLA match, and stem cell source lost significance if the donor was 12 years old, but increased in magnitude and was significant if the donor was ≥12 years old (OR, 13.6; 95% CI, 2.8 to 39.6). Patient age was not a significant risk factor in multivariate analyses.
Population sample inclusion criteria did not consider important factors as:
donor history of blood transfusions and the wide age range of participants. Age was used as a surrogate for sexual-naivety and therefore it does not directly answer the question.
The study concluded that when selecting among sibling donors for a pediatric patient, priority should be given to donors 12 years of age or younger and that selection can be done independently of donor gender and sex match. Other explanations for the results of this study cannot be excluded and many limitations on patient enrolment criteria, age, and confounding factors could have impacted the results.

Oncogenic mutations combined with MRD improve outcome prediction in pediatric T-cell ALL

Risk stratification for pediatric T-ALL is based primarily on clinical findings and MRD. This study aimed to identify new genetic prognostic factors to improve the detection of patients at risk of relapse. Mutations in the Notch1 and Ras pathway were selected based on their recognition as oncogenic pathways in T-ALL and the reports in adult literature demonstrating their prognostic value.
220 patients treated prospectively on the FRALLE 2000T study (France) from 2000 to 2010 who had DNA material available were retrospectively analyzed for mutations in Notch1 (N), FBXW7 (F), K-RAS/N-RAS (R) and PTEN (P), both somatic and germline. The low-risk group was defined as having N/F mutations in the absence of R/P mutations. High-risk group was defined as having both N/F and R/P somatic mutations or either N/F germline or R/P germline mutations. Multi-variable regression analyses were performed to assess whether this classification of patients into low (n=111) and high-risk (n=109) groups was prognostic. Of note, on the FRALLE 2000T study, risk stratification was based on MRD.
Classification into low-risk and high-risk genetic groups was an independent prognostic risk factor. 5-year cumulative incidences of relapse were 11% and 36% for low-risk and high-risk groups respectively. When combined with WBC count and MRD, genetic risk groups helped further identify patients at low and high risk of relapse: Patients with a WBC > 200,000/µL, high-risk genetics and MRD > 1x10e4 had a cumulative incidence of relapse of 46% as compared to only 2% for patients with a WBC 200,000/µL, low-risk genetics and MRD 1x10e4.
Retrospective analysis of a prospective cohort. Similar study based on UK2003 study did not demonstrate significance of K/N-ras and PTEN mutations.
The combination of white cell count > 200,000/µL, MRD positivity, and genetic risk group were helpful in stratifying relapse risk in pediatric T-ALL, in particular identifying the low-risk group, in this cohort. These findings are in contrast with the findings from the UKALL2003 data which did not find genetic risk factors contributory (https://www.ncbi.nlm.nih.gov/pubmed/26220040).

IKZF1plus Defines a New Minimal Residual Disease-Dependent Very-Poor Prognostic Profile in Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia.

This paper from the International BFM Study Group further defines the IKZF1 gene as a very poor prognostic marker in pediatric B-cell precursor ALL. The study group looked to refine the prognostic strength of the IKZF1 deletion by looking at the effect of the co-occurring gene deletions.
The study analysed 991 patients with B-cell precursor ALL from the European group (AIEOP-BFM) trial with complete information for copy number alterations of major genes associated with outcome in ALL. There was also a smaller replication cohort of 417 patients from the same trial.
The study then analysed gene combinations and evaluated patient outcomes including how gene combinations affected outcomes combined with MRD status.
"IKZF1plus" was defined as an IKZF1 deletion co-occurring with deletions in CDKN2A, CDKN2B, PAX5 or PAR1 in the absence of a deletion in the ERG gene.
The IKZF1plus group made up of 6% of patients in the cohort of children with B-cell precursor ALL (n=63). The 5-year EFS for this IKZF1plus group was 53% compared to 79% for those with lone IKZF1 deletions and 87% for patients who lacked the IKZF1 deletion altogether. The 5-year EFS when combining the IKZF1plus combination with MRD was 95% for standard risk MRD versus 40% for intermediate MRD and 30% for high-risk MRD.
Given differences in therapy and timing/method of MRD measurement it's not clear whether these results can be generalized to other study groups.
The use of the IKZF1 deletion in combination with specific additional single gene deletions – the so-called IKZF1plus – provides an independent and strong molecular stratification marker in addition to MRD measurements. The IKZF1plus group with positive MRD are a particularly high-risk group and should be assigned additional/experimental treatments, which will be evaluated in the upcoming AIEOP-BFM ALL 2017 trial. On the other hand, the IKZF1plus genotype loses prognostic significance when MRD is negative.

Genotype-Specific Minimal Residual Disease Interpretation Improves Stratification in Pediatric Acute Lymphoblastic Leukemia

Cytogenetic analysis and minimal residual disease (MRD) assessment are used to personalize treatment in current protocols for acute lymphoblastic leukemia (ALL). MRD status is currently assigned using cut-off limits in treatment protocols. This study assessed MRD as a continuous variable using PCR-based MRD assessment (Ig/TCR rearrangements) and correlated results with somatic genetic changes.
3,113 consecutive patients with ALL treated in the MRC UKALL2003 protocol (2003 to 2011) were eligible and 2,542 were analysed. Patients were classified into four mutually exclusive cytogenetic genetic groups: good risk: ETV6-RUNX1, high hyperdiploidy (51 to 65 chromosomes); high risk: KMT2A (MLL) fusions, near haploidy, low hypodiploidy ( 40 chromosomes), iAMP21, and TCF3-HLF; intermediate risk: TCF3-PBX1 and all other patient-cases with B-ALL; and patients with T-ALL. MRD at end of induction was assessed as a continuous variable with a minimum detection level of 1x10e-5.
MRD results were highly dependent on genetic risk groups with ETV6-RUNX1 but also TCF3-PBX1 showing rapid MRD-clearance (in 36% and 43% respectively). Patients with iAMP21 on the other hand had a high rate of recurrence even in those with negative MRDs. T-ALL patients more frequently had not reportable results which renders the Ig/TCR PCR-based technique less reliable for this patient group. In T-ALL, outcomes were associated with MRD-negativity or frank positivity (>=5%) but not intermediate results.
This is a post hoc analysis of MRD results and treatment was not based on these findings. The impact of continuous MRD assessment was therefore not assessed on possible treatment modifications.
As previously known, PCR-based high-sensitive MRD-assessment is a powerful tool and results differ between different cytogenetic groups. The inclusion of iAMP21 as a high-risk somatic change per se was supported.

Reduced-Intensity Delayed Intensification in Standard-Risk Pediatric Acute Lymphoblastic Leukemia Defined by Undetectable Minimal Residual Disease: Results of an International Randomized Trial (AIEOP-BFM ALL 2000)

Delayed intensification (DI) in acute lymphoblastic leukemia (ALL) is an essential element in treatment protocols but is intensive and associated with toxicity. This study originated from the AIEOP-BFM 2000 trial and aimed at testing non-inferiority of reduction in DI from 49 days (P-II) to 29 days (P-III) with a shorter duration of steroids and lower doses of cyclophosphamide, vincristine, and doxorubicin.
The randomized prospective trial AIEOP-BFM ALL 2000 is a European multinational cohort study. From 2000 to 2006, 4,937 patients were enrolled on the trial. Of 1,346 patients with standard risk criteria (SR, the lowest risk category), 1,164 were randomly assigned on the reduced regimen P-III or the standard regimen P-II (roughly half in each arm).
Median follow-up time was 8.4 years. The disease-free survival was 91.8% vs. 95.8% with the reduced regimen vs. the standard treatment with 62 versus 42 events (p=0.04). 8-year OS was 96.1% versus 98% (NS). Acute toxicity was about the same in the two regimens with slightly more life-threatening events occurring with standard therapy P-II (n=10 vs. 7). The reduced regimen P-III was not associated with similar outcomes compared to the standard regimen P-II and therefore, treatment reduction in SR patients with ALL was not successful in this trial. The only subgroups with similar outcomes were ETV6-RUNX1 positive ALL and patients aged 1 to 6 years.
The results of this trial are specific to the BFM backbone and risk stratification which differs compared to other large international trials.
Treatment reduction in delayed intensification was not successful in this trial for Standard risk ALL patients. ETV6-RUNX1 status was added as a favorable cytogenetic marker in the AIEOP-BFM 2009 trial. The 2009 trial will ask whether a reduction of the 4-drug induction by using half the daunorubicin dose in low-risk patients leads to comparable outcomes.