Precursor Lymphoblastic Leukemia Research Articles

A Comparative Analysis of Early T-Cell Precursor Lymphoblastic Leukemia/Lymphoma (ETP-ALL/LBL) and Non-ETP-ALL/LBL in a Tertiary Cancer Care Center Based in Western India.

Early T-cell precursor lymphoblastic leukemia (ETP-ALL) has a unique immunophenotype with very early T-cell differentiation. The current study summarises the distinct clinicopathological aspects of ETP-ALL and compares them with non-ETP-ALL. Twenty-nine ETP-ALL and 191 non-ETP-ALL cases were retrieved between 2018 and 2021. A P value was determined for each of the patient charaterisics (Table 1) to see for any significant relationship (P < 0.05) with ETP-ALL versus non-ETP-ALL. Kaplan-Meier log rank test was applied to look for any significant differences in OS for both the ALLs. ETP-ALL had an incidence of 12.6% out of total T-Acute lymphoblastic leukemia (T-ALL/LBL) in the past 3-years. Compared to non-ETP-ALL, ETP-ALL cases were associated with lower median age and male-to-female ratio. There was no statistically significant difference in the complete remission rate between both the subtypes. ETP-ALL was seen to be associated with high induction failure and relapse rate compared to non-ETP-ALL. To summarise, since the 2-year OS was poor compared to western research (for both ALLs), an intensive chemo-regimen should be implemented in the current situation. Some unusual markers were observed on flow-cytometry (ETP-ALL), which can be useful for MRD quantification, prognosis, and further trials for newer targeted therapies.

EPID-14. EPIDEMIOLOGICAL SURVEY OF RADIATION-INDUCED MENINGIOMA: A SEER ANALYSIS

Abstract OBJECTIVE Radiation-induced meningioma (RIM) is a rare but serious consequence of cranial and cranio-spinal external beam radiation therapy (EBRT). While recent studies have characterized the epidemiological features of RIM in small populations or systematic reviews, none have studied RIM within a national database for a more generalizable description of this disease entity. The objective of this study was to identify and characterize the population with RIMs within a large national database. METHODS From the SEER Research Plus Nov 2020 (1975-2018) dataset, 56161 patients treated with EBRT for the following indications were identified: ocular/orbital tumors, brain/nervous system tumors, and leukemias. Next, a sub-population of 185 RIM patients, defined by histologically distinct precedent tumor treated with EBRT and a minimum 5-year latency from EBRT to meningioma, was identified. RIM prevalence, latency, pathologic behavior, and multiplicity were determined, and comparisons were made by sex, race, and original indication for EBRT. RESULTS The prevalence of RIM among the EBRT population was 0.33%. Among RIM patients, 41.62% were male and 84.32% were Caucasian. Average latency to RIM development was 23.39 ± 8.65 years, with no significant difference between sexes or races. Fourteen patients had multiple RIMs at diagnosis, while four patients experienced recurrent RIM. RIMs classified as “malignant” had a shorter latency (16.30 years) than “borderline” (26.11 years) or “benign” (23.36 years) RIMs. Of the most common original EBRT indications, precursor lymphoblastic leukemia (29.89 years) had longer latency to RIM than medulloblastoma (21.57 years) or astrocytoma (22.67 years); this may be explained by lower doses of radiation used for lymphoblastic leukemia treatment. CONCLUSION These data provide novel epidemiological information—most importantly, prevalence—of RIM in a nationwide population, and they suggest that RIM occurs in patients independent of sex or race. These findings will aid clinicians in explaining the risk of RIM development to patients undergoing EBRT.

Chromothripsis is a frequent event and underlies typical genetic changes in early T-cell precursor lymphoblastic leukemia in adults

Chromothripsis is a mitotic catastrophe that arises from multiple double strand breaks and incorrect re-joining of one or a few chromosomes. We report on incidence, distribution, and features of chromothriptic events in T-cell acute lymphoblastic leukemias (T-ALL). SNP array was performed in 103 T-ALL (39 ETP/near ETP, 59 non-ETP, and 5 with unknown stage of differentiation), including 38 children and 65 adults. Chromothripsis was detected in 11.6% of all T-ALL and occurred only in adult cases with an immature phenotype (12/39 cases; 30%). It affected 1 to 4 chromosomes, and recurrently involved chromosomes 1, 6, 7, and 17. Abnormalities of genes typically associated with T-ALL were found at breakpoints of chromothripsis. In addition, it gave rise to new/rare alterations, such as, the SFPQ::ZFP36L2 fusion, reported in pediatric T-ALL, deletions of putative suppressors, such as IKZF2 and CSMD1, and amplification of the BCL2 gene. Compared to negative cases, chromothripsis positive T-ALL had a significantly higher level of MYCN expression, and a significant downregulation of RGCC, which is typically induced by TP53 in response to DNA damage. Furthermore we identified mutations and/or deletions of DNA repair/genome stability genes in all cases, and an association with NUP214 rearrangements in 33% of cases.

Relevance of flow cytometric categorization and end-of-induction measurable residual disease assessment in pediatric and adult T-lymphoblastic leukemia patients.

BackgroundT-lymphoblastic leukemia (T-ALL) patients expressing myeloid/stem cell antigens are classified as early T-cell precursor lymphoblastic leukemia (ETP-ALL) or near-ETP-ALL.MethodsClinico-laboratory profiles, flow cytometric end-of-induction measurable residual disease (EOI-MRD), and survival of treatment naïve T-ALL patients were analyzed according to their immunophenotypic subtypes.ResultsAmong 81 consecutive T-ALL patients diagnosed, 21% (N=17) were ETP-ALL and 19% (N=15) were near-ETP-ALL. EOI-MRD was detectable in 39% of the 59 samples tested (31.6% of pediatric samples and 52.4% of adult samples). The frequency of EOI-MRD positivity was significantly higher among ETP-ALL (75%, P=0.001) and near-ETP-ALL (71%, P=0.009) patients compared to that in conventional-T-ALL (con-T-ALL) patients (22.5%). CD8 (P=0.046) and CD38 (P=0.046) expressions were significantly upregulated in the EOI blasts of con-T-ALL and ETP-ALL samples, respectively. The 2-year rates of overall (OS), relapse-free (RFS), and event-free survival (EFS) among the T-ALL patients (pediatric vs. adult) was 79.5% vs. 39.8% (P<0.001), 84.3% vs. 60.4% (P=0.026), and 80.3% vs. 38% (P<0.001), respectively. Univariate analysis revealed that 2-year EFS and RFS of pediatric T-ALL patients was independent of T-ALL subtype and was influenced only by EOI-MRD status. However, 2-year OS, RFS, and EFS among adult T-ALL patients were EOI-MRD independent and influenced only by the near-ETP-ALL phenotype.ConclusionTwo-year survival among pediatric and adult T-ALL patients is attributed to EOI-MRD status and near-ETP-ALL phenotype, respectively.

How I Diagnose Acute Leukemia of Ambiguous Lineage.

Classification of acute leukemia involves assigning lineage by resemblance to normal progenitor cells. This approach provides descriptive information about the blast cells that is useful for disease monitoring, provides clues to pathogenesis, and can help clinicians select effective chemotherapeutic regimens. Acute leukemias of ambiguous lineage (ALALs) are those leukemias that either fail to show evidence of myeloid, B-, or T-lymphoid lineage commitment or show evidence of commitment to more than 1 lineage. The different treatment regimens for acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) make ALAL a challenge both diagnostically and therapeutically. Current classification criteria have reduced the reported incidence of mixed-lineage leukemias by emphasizing fewer markers and categorizing some biphenotypic leukemias with recurrent cytogenetic abnormalities as other entities. Several recent studies have explored the genomic and epigenetic landscape of mixed-phenotype acute leukemia (MPAL) and have suggested a further refinement of the World Health Organization classification to emphasize the genomic heterogeneity of MPAL. Genomic and expression profile data for MPAL reveal mutations commonly seen in both AML and ALL, with T-/myeloid MPAL showing overlapping features with early T-cell precursor lymphoblastic leukemia. Our review aimed to discuss the diagnostic challenges, recent genomic studies, and therapeutic strategies in this poorly understood disease.

Aberrant Expression of CD19 in Early T-cell Precursor Lymphoblastic Leukemia- A Diagnostic Challenge

A systematic approach is required to diagnose acute leukemia. Most of the cases are satisfactorily diagnosed and categorized into subtypes. However, a few cases pose diagnostic dilemma secondary to immunophenotypic aberrancies which are defined as antigens that are normally restricted to a different lineage and expressed by a neoplastic population while absent from its normal non neoplastic counterpart. We report a rare case of Early T-cell Precursor Lymphoblastic Leukemia with aberrant expression of CD19. A 7-year-old boy referred to our hospital with his cervical lymph node biopsy reported as lymphoproliferative disorder. The patient was COVID-19 positive. Chest X-ray showed mild right sided pleural effusion with huge mediastinal mass. Flow cytometry on peripheral blood used to establish the diagnosis. The case is reported to improve knowledge regarding aberrant expression of markers. Hematopathology teams should be aware of this phenomenon so that appropriate workup can be done to reach correct diagnosis.

Expression of CD20 Affects Early MRD Response to Rituximab in Adult B Cell Precursor Lymphoblastic Leukemia of the GMALL 08/2013 Trial

Expression of CD20 Affects Early MRD Response to Rituximab in Adult B Cell Precursor Lymphoblastic Leukemia of the GMALL 08/2013 Trial

Blast phase of chronic myeloid leukemia presenting as early T-cell precursor lymphoblastic leukemia.

Blast phase of chronic myeloid leukemia presenting as early T-cell precursor lymphoblastic leukemia.

A Preliminary Study of FTIR Spectroscopy as a Potential Non-Invasive Screening Tool for Pediatric Precursor B Lymphoblastic Leukemia.

Early detection of the most common pediatric neoplasm, B-cell precursor lymphoblastic leukemia (BCP-ALL), is challenging and requires invasive bone marrow biopsies. The purpose of this study was to establish new biomarkers for early screening to detect pediatric leukemia. In this small cohort study, Fourier transform infrared (FTIR) spectra were obtained from blood sera of 10 patients with BCP-ALL and were compared with the control samples from 10 children with some conditions other than neoplasm. Using various analytical approaches, including a new physical model, some significant differences were observable. The most important include: the different peak area ratio 2965/1645 cm−1 (p = 0.002); the lower average percentage of both β-sheet and β-turn protein structures in the sera of BCP-ALL patients (p = 0.03); an AdaBoost-based predictive model for classifying healthy vs. BCP-ALL patients with 85% accuracy; and the phase shift of the first derivative in the spectral range 1050–1042 cm−1 correlating with white blood cell (WBC) and blast cell count in BCP-ALL patients contrary to the samples obtained from healthy controls. Although verification in larger groups of patients will be necessary, these promising results suggest that FTIR spectroscopy may have future potential for the early screening of BCP-ALL.

Treatment and Prognosis of Adult T Cell Acute Lymphoblastic Leukemia

To analyze the treatment and prognosis of T cell acute lymphoblastic leukemia(T-ALL)in adults. Method The clinicobiogical and survival data of 68 adult patients with newly diagnosis T-ALL were retrospectively analzyed. Results The median age of these 68 patients was 23 years(14-60 years).T-ALL was more common in men(81%).After the first cycle of treatment,complete remission was achieved in 50 patients(73%).The highest complete remission(CR) rate was in patients with cortex T-ALL(100%),followed by other T-ALL(73%)and early T-cell precursor lymphoblastic leukemia(54%),(χ 2=5.712,P=0.058).The CR rate for adults aged >35 years was significantly lower than that of patients aged ≤ 35 years(40% vs. 79%,χ 2=6.364,P=0.012).The overall CR rate after the second treatment course was 93%.For patients treated with chemotherapy,autograft hematopoietic stem cell transplantation(auto-SCT),and allogeneic SCT,the median relapse free survival was 10 months,24 months,and not reached,respectively(P=0.002).The 5-year overall survival rate was 25% for all patients;for patients treated with chemotherapy,auto-SCT and allogeneic SCT,the median overall survival was 24 months,34 months,and 30 months,respectively(P=0.007),and the 5-year overall survival rate was 9%,33%,and 38%(P=0.037).Multivariate analysis showed leukocyte count ≥100×10 9/L was a risk factor for decreased relapse free survival(risk ratio 2.540,95%CI=1.058-6.099,P=0.037). Conclusion Adult T-ALL patients have poor prognosis,which may be improved by SCT.

Significance of Neuropilin-1 Expression, Ceruloplasmin oxidase and Copper in Egyptian Children with B-Lineage Acute Lymphoblastic Leukemia

Background: Neuropilins are transmembrane glycoproteins that act as receptors for vascular endothelial growth factors (VEGF) and are involved in the process of tumor angiogenesis. Ceruloplasmin is a member of the multi copper oxidase family. It has antioxidant properties that play a central role in protection of the body against advanced oxidation protein products. Objective: This study was aimed to assess the expression of Neuropilin-1(NRP-1) on blasts of B-lineage precursor lymphoblastic leukemia (ALL) to be used in the diagnostic panel of this disease. We also aimed to assess the alteration of the levels of ceruloplasmin oxidase and copper as a compensatory mechanism to minimize the effects of reactive oxygen species resulting from leukemias. Subjects and Methods: This study was conducted on 40 children with newly diagnosed B-lineage precursor lymphoblastic leukemia. 40 age-matched controls were enrolled to serve as control. The expression of NRP-1 on peripheral blood samples was evaluated by flow cytometry as the proportion of positive cells expressing the marker. Ceruloplasmin oxidase and copper levels were assessed by immunoturbidimetric assay. Results: There was highly significant increase in the proportion of positivity of NRP -1 in patients compared with control group (P<0.001) Ceruloplasmin oxidase and copper levels were also higher in patients compared with control group (P<0.001). Conclusions: It could be concluded that NRP-1 is a valuable marker for diagnosis of B-lineage ALL. There is an increase in the levels of ceruloplasmin oxidase and copper which at the time of diagnosis of B-ALL.

Significance of Neuropilin-1 Expression, Ceruloplasmin oxidase and Copper in Egyptian Children with B-Lineage Acute Lymphoblastic Leukemia

Background: Neuropilins are transmembrane glycoproteins that act as receptors for vascular endothelial growth factors (VEGF) and are involved in the process of tumor angiogenesis. Ceruloplasmin is a member of the multi copper oxidase family. It has antioxidant properties that play a central role in protection of the body against advanced oxidation protein products. Objective: This study was aimed to assess the expression of Neuropilin-1(NRP-1) on blasts of B-lineage precursor lymphoblastic leukemia (ALL) to be used in the diagnostic panel of this disease. We also aimed to assess the alteration of the levels of ceruloplasmin oxidase and copper as a compensatory mechanism to minimize the effects of reactive oxygen species resulting from leukemias. Subjects and Methods: This study was conducted on 40 children with newly diagnosed B-lineage precursor lymphoblastic leukemia. 40 age-matched controls were enrolled to serve as control. The expression of NRP-1 on peripheral blood samples was evaluated by flow cytometry as the proportion of positive cells expressing the marker. Ceruloplasmin oxidase and copper levels were assessed by immunoturbidimetric assay. Results: There was highly significant increase in the proportion of positivity of NRP -1 in patients compared with control group (P<0.001) Ceruloplasmin oxidase and copper levels were also higher in patients compared with control group (P<0.001). Conclusions: It could be concluded that NRP-1 is a valuable marker for diagnosis of B-lineage ALL. There is an increase in the levels of ceruloplasmin oxidase and copper which at the time of diagnosis of B-ALL.

T-lymphoid/myeloid mixed phenotype acute leukemia and early T-cell precursor lymphoblastic leukemia similarities with NOTCH1 mutation as a good prognostic factor.

Purpose: T-lymphoid/Myeloid Mixed phenotype acute leukemia (T/M-MPAL) is ambiguous leukemia which overlaps with early T-cell precursor lymphoblastic leukemia (ETP-ALL). We have revisited the immunophenotyping profile of T/M-MPAL and ETP-ALL to identify differences and/or similarities, as these entities represent a therapeutic challenge in clinical practice.Patients and methods: A total of 26 ETP-ALL and 10 T/M-MPAL cases were identified among 857 cases of childhood leukemia (T-ALL, n=266 and AML, n=591) before any treatment decisions. The variables analyzed were age strata, sex, clinical features, immunophenotyping, and molecular aberrations. Immunophenotyping was performed in all samples using a panel of cytoplasm and membrane antibodies to identify the lineage and blast differentiation. The mutational status of STIL-TAL1, TLX3, RUNX1, NOTCH1, FBXW7, FLT3, IL7R, RAS, KTM2A, and CDKN2A/B was tested using RT-PCR, FISH, and PCR sequencing methods. The outcomes were assessed in terms of overall survival (OS).Results: The immunophenotypes were similar in ETP-ALL and T/M-MPAL, regarding the cellular expression of CD34, CD117, CD13/CD33, and CD11b, although CD2 and HLA-DR were more frequent in T/M-MPAL (p<0.01). aMPO positivity and myelomonocyte differentiation were definitive in separating both entities. NOTCH1, FLT3-ITD, and N/KRAS mutations as well as TLX3 and KMT2A rearrangements were found in both ETP-ALL and T/M-MPAL. Thirty-one patients received ALL protocol whereas five had AML therapy. The overall 5-year survival rate (pOS) was 56.4% for patients treated using ALL protocols. No differences were observed between T/M-MPAL (pOS of 57%) and ETP-ALL (pOS of 56%) patients. The prognostic value of NOTCH1mut was associated with significantly better OS (pOS 90%) than NOTCH1wt (pOS 37%) (p=0.017).Conclusion: This research can potentially contribute to NOTCH1 as targeted therapy and prognostic assessment of T-cell mixed phenotype leukemia.

CD9 predicts ETV6-RUNX1 in childhood B-cell precursor acute lymphoblastic leukemia

IntroductionThe ETV6-RUNX1 is a fusion gene associated with a good outcome in B-cell precursor lymphoblastic leukemia. ObjectiveThis study aimed to re-evaluate the CD9 cellular expression by flow cytometry (FC) as a possible tool to predict the presence of ETV6-RUNX1. MethodChildhood B-cell precursor lymphoblastic leukemia cases were included (n=186). The percentage of CD9-labeled cells and the median fluorescence intensity ratio were used for correlation with the molecular tests. Receiver Operating Characteristic curves were performed to determine the likelihood of the CD9 expression predicting ETV6-RUNX1. ResultsThe ETV6-RUNX1 was found in 44/186 (23.6%) cases. Data analysis revealed that the best cutoff for CD9 percentage was 64%, with an accuracy of 0.84, whereas the best cutoff for CD9 median fluorescence intensity ratio was 12.52, with an accuracy of 0.80. A strong association was observed between the level of CD9 expression and the presence of ETV6-RUNX1. ConclusionThese data confirm that the CD9 expression could be used for risk stratification in clinical practice.

A Novel Oral Histone Deacetylase Inhibitor Chidamide Is Highly Effective and Well-Tolerated in Adult Early T-Cell Precursor and Ph-like Acute Lymphoblastic Leukemia

Background Early T-cell precursor (ETP) lymphoblastic leukemia (ETP-ALL) is a neoplasm with an unique T-cell immunophenotype indicating limited early T differentiation. Ph-like ALL is a B-cell neoplasm which lack BCR-ABL1 translocation but similar expression-pattern of the BCR-ABL1-positive ALL. The optimal therapeutic approaches for ETP-ALL and Ph-like ALL are poorly characterized. Chidamide is a novel and orally active benzamide class of histone deacetylase inhibitor (HDACi) and approved for peripheral T-cell lymphoma (PTCL).Methods Based on the pediatric-inspired, PEG-L-asparaginase-intensified and MRD-directed PDT-ALL-2016 protocol, we designed two open-label, one-arm, multi-site trials, PDT-ETP-ALL (NCT03553238) and PDT-Ph-Like (NCT03564470), to evaluate the safety and effect of HDACi chidamide for adult ETP-ALL and Ph-Like-ALL group, respectively. The protocols were approved by Institutional Review Broad (IRB). Chidamide at a dose of 10mg/day will be added to ETP-ALL group from induction therapy to consolidation therapy according to PDT-ETP-ALL protocol. Chidamide and dasatinib will be added to HDACi cohort and TKI cohort, respectively, based on cytogenetics and next-generation-sequencing classification, according to PDT-Ph-Like protocol. Primary study endpoint is event-free survival and secondary study endpoints are complete remission (CR) and MRD after induction, adverse event and overall survival.Result Between FEB 2016 to DEC 2017, 24 patients with ETP-ALL were enrolled into PDT-ETP-ALL trial, 4 female patients and 20 male patients, a median age 22 years old (range, 14-22 years old). A total of 33 patients with Ph-like ALL has been enrolled into PDT-Ph-Like trial, 16 female patients and 17 male patients, a median age of 23 years old (range, 14-55 years old) 11 patients in TKI arm and 22 patients in HDACi arm. Ph-like ALL with CRLF2 high-expression, CRLF2/EPO/JAK2 rearrangement, JAK/STAT/IL-7R/SH2B3 mutation, will be assigned to HDACi arm. Targeted next-generation sequencing revealed ETP-ALL patients harbor high rates of mutations in factors involved in cytokine and JAK/STAT signaling pathway (62%), epigenetic regulation (52%) and hematopoietic development (35%). At the same time, we also performed NGS assessment with the same panel of Ph-like ALL patients. Of note, ETP-ALL and Ph-like ALL share the mutations involved in JAK/STAT signaling pathway (JAK1, JAK2, IL-7R) and histone modification (SETD2, KMT2A, EZH2, KMT2C), which might indicate the underlying mechanism of sensitivity of ETP-ALL and Ph-like ALL to HDACi chidamide. Chidadmide was well-tolerated in ETP-ALL and Ph-like ALL patients. Fatigue, nausea, vomit, neutropenia and thrombocytopenia are common chidamide-associated adverse events (AE) with Common Terminology Criteria for Adverse Events (CTCAE) grade I-II. Complete remission and Flow-MRD-negative rate after induction therapy for ETP-ALL and Ph-Like-ALL were 87% and 67%, 77% and 60%, respectively. Six patients with ETP-ALL (25%, 6/24) underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT), and 11 patients with Ph-like ALL received allo-HSCT. With a median follow-up of 20 months (range, 7-31 months), estimated 2-year event-free-survival (EFS) of ETP-ALL and Ph-like ALL is 83%, 70%, respectively.Conclusion: Our preliminary data of PDT-ETP-ALL and PDT-Ph-like ALL trials suggest that a novel HDACi chidamide is effective and well-tolerated in adult ETP-ALL and Ph-like ALL, which deserve further extended clinical trial. DisclosuresZhou:CHIPSCREEN: Consultancy. Carter:novartis: Research Funding; AstraZeneca: Research Funding.

CD20 Expression and Response to Rituximab Treatment in B-Cell Precursor Lymphoblastic Leukemia - Results of the GMALL 08/2013 Trial

Introduction: Treatment with Rituximab has improved the outcome for patients with B-cell precursor ALL (B-ALL) and is conventionally restricted to patients with CD20 expression on leukemic blasts above a level of 20%. However, it is not known whether this arbitrary cutoff is biologically meaningful, or if also patients with lower CD20 expression levels profit from Rituximab treatment. In addition, CD20 expression levels might differ between different compartments and can change during early treatment. Therefore, in the current German Multicentric Acute Lymphoblastic Leukemia (GMALL) 08/2013 trial Rituximab is applied to all BCR-ABL1-negative B-ALL patients during induction and early consolidation irrespective of the initial blast CD20 expression. In this study, CD20 expression on blasts at diagnosis and after a 5-day dexamethasone prephase is thoroughly characterized and correlated to MRD response after induction I including one dose of Rituximab to evaluate its impact on response to Rituximab.Methods: Comparative quantification of CD20 expression levels was performed at diagnosis (blood and bone marrow (BM)) and at the end of the prephase (blood) and correlated to minimal residual disease (MRD) response after early Rituximab treatment. Samples were subjected to the EuroFlow standardized stain, lyse and wash and instrument setting procedures (van Dongen et al., 2012) and stained with a 3-tube, 8-color panel, containing the markers CD45, CD20, CD10, CD66c, CD3, CD19, CD71, CD9, CD13+33, CD34, CD22, CD11a, and CD38. CD20 median fluorescence intensities (CD20-MFI) as well as percentages of CD20+ B-ALL blasts among all blasts (%CD20+) were measured. MRD was quantified at day 22 after induction I using real-time quantitative PCR of clonal immune gene rearrangements. MRD response after induction was defined as MRD decrease to a level below 10-4, MRD persistence as detection of quantifiable MRD ≥10-4.Results: FCM results of a total of 166 samples of 84 B-ALL patients were evaluated. CD20 expression of circulating blasts significantly increased after a 5-day prephase in common/pre-B-ALL (c/pre-B) but not in pro-B-ALL in a cohort of 36 paired peripheral blood samples (Fig. 1 A-B, n=8 pro-B-ALL, paired t-test of CD20-MFI and %CD20: p=.1016 and p=.1660, Fig. 2 A-B, n=28 c/pre-B-ALL, p=.0029 and p=0.0060). Interestingly, the increase of CD20 expression on B-ALL blasts contrasted that on mature normal B-cells, where a decrease occurred in pro-B-ALL and c/pre-B-ALL samples at day 6 (paired t-test: p=.0485 and p<.0001, respectively) (Fig.1A, 2A).In 24 paired blood/BM pre-treatment samples the percentage of CD20+ blasts was significantly higher in blood in c/pre-B-ALL (n=19: paired t-test: p=.0009), but not in pro-B-ALL (n=5: paired t-test: p=.095) (Fig. 1C, 2C). The significance levels were not consistently reached for the comparison of CD20-MFI. Nevertheless, we show that in 3/24 (12.5%) patients the percentage of CD20+ B-ALL blasts in BM did not reach the arbitrary cutoff of 20% whereas the matching diagnostic blood did.Finally, CD20 expression levels from 25 BM and 30 pre-treatment blood samples and 38 blood samples after dexamethasone prephase from 54 patients were assessed (in total 93 samples). In further calculations we correlated MRD response values after one dose of Rituximab with the CD20 expression levels on blasts. In detail, the highest measured percentage value of %CD20 per patient in the group of MRD responders was compared to the highest measured %CD20 per patient in the group of MRD persisters. CD20 expression levels were significantly lower in 33 MRD persisters compared to 21 MRD responders (Fig 3, p=.0336). The difference was mainly attributable to a high frequency of MRD persisters in pro-B-ALL compared to c/pre-B-ALL (MRD persistence in pro-B-ALL 8/9 (89%) compared to 25/45 (56%) in c/pre-B-ALL).Conclusion: We measured significant differences in CD20 expression levels on leukemic blasts (i) between pre-treatment blood and BM and (ii) between pre-treatment blood and blood after prephase in patients with c/pre-B-ALL challenging the conventional eligibility criteria for the CD20 targeted treatment. MRD responders tended to have higher CD20 expression levels compared to MRD persisters. Extension of the patient cohort is ongoing to confirm this observation. Furthermore, we will analyze the impact of 3 rituximab applications after induction phase II. [Display omitted] DisclosuresRitgen:abbvie: Research Funding; Roche: Honoraria, Research Funding. Viardot:Gilead Kite: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Amgen: Consultancy. Kneba:Roche: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria. Goekbuget:Kite / Gilead: Consultancy; Celgene: Consultancy; Novartis: Consultancy, Other: Travel support, Research Funding; Pfizer: Consultancy, Other: Travel support, Research Funding; Amgen: Consultancy, Other: Travel support, Research Funding. Brüggemann:Incyte: Consultancy; Amgen: Consultancy, Research Funding, Speakers Bureau; PRMA: Consultancy; Affimed: Research Funding; Regeneron: Research Funding; Pfizer: Speakers Bureau; Roche: Speakers Bureau.

T-cell transcription factor GATA-3 is an immunophenotypic marker of acute leukemias with T-cell differentiation

T-cell transcription factor GATA-3, known to play a role in early T-cell development and in the development of T-cell neoplasms, is expressed at high levels in fetal and adult thymus, as well as in acute leukemias with T-cell differentiation, including T-lymphoblastic leukemia/lymphoma (22/22 cases), early T-cell precursor lymphoblastic leukemia (11/11 cases), and mixed-phenotype acute leukemia, T/myeloid (4/5 cases), but only rarely in acute myeloid leukemia/myeloid sarcoma (1/36 cases), and not in B-lymphoblastic leukemia (0/16 cases). In contrast, T-bet, the other T-cell transcription factor that controls Th1/Th2 T-cell fate, is not expressed to any significant extent in immature thymocytes or in cases of T-lymphoblastic leukemia or acute myeloid leukemia/myeloid sarcoma, but is expressed in most cases (15/16) of B-lymphoblastic leukemia and in mixed-phenotype acute leukemia, B/myeloid. GATA-3-positive acute leukemias with T-cell differentiation were also found to express proto-oncogene C-MYC, in an average of 52% of neoplastic cells, which, along with GATA-3, may contribute to leukemogenesis, as suggested by transgenic mouse models. We conclude that GATA-3 is a sensitive and specific marker for the diagnosis of acute leukemias with T-cell differentiation and may be a useful addition to the panel of immunophenotypic markers for the diagnostic evaluation of acute leukemias.

P021 Development of laboratory-developed chimerism analysis assay targeting t lymphocytes in hematopoietic stem cell transplantation

Aim Hematopoietic stem cells transplantation (HSCT) is one of the gold standard treatment for the patients with hematologic malignancies. After the transplantations, the engraftment of the stem cells is monitored using various methods. Short tandem repeats (STR) are one of the sensitive markers for chimerism. We designed laboratory-developed chimerism analysis assay using 8 short tandem repeat regions. Methods Total 38 patients who received allogeneic HSCT were involved in this study. The diagnosis of the patients included acute myeloid leukemia, precursor lymphoblastic leukemia, myelodysplastic syndrome, hemophagocytic lymphohistiocytosis, chronic eosinophilic leukemia, paroxysmal nocturnal hemoglobinuria, juvenile myelomonocytic leukemia, plasma cell myeloma, and congenital neutropenia. DNA was extracted from both ACD anticoagulated blood and T lymphocytes isolated by EasySep (STEMCELL Technologies, Vancouver, Canada). We designed the assays, named LDT 8, targeting D3S1358, D5S818, TH01, D7S820, D12S391, FGA, D18S51, and Penta E. The amplicons were analyzed using Performance Optimized Polymer 7 (Applied Biosystems Norwalk, USA) by 3130 xl genetic analyzer (Applied Biosystems). The results LDT8 were compared with commercially available PowerPlex 21 (Promega, Madison, USA) and Mentype 13 (Biotype, Dresden, Germany). In addition, we also compared the chimerism status of whole blood to that of T lymphocyte. Results Compared with PowerPlex 21 and Mentype 13 respectively, 90.1% and 84.9% of specimens showed less than 5%p in overall discrepancies. In the comparison between whole blood and T lymphocytes, most of specimens showed the higher recipient proportion of DNA in T lymphocytes. Conclusions We developed the economical and effective chimerism analysis assay using 8 short tandem repeat regions. Also, we identified discrepancies among two different cell sources. Further study for long-term monitoring of chimerism status in different cell subsets would be helpful in patients who received hematopoietic stem cell transplantation.

Anterior Chest Wall Bulging: A Rare Initial Manifestation of Acute Lymphoblastic Leukemia in a Child

Acute lymphoblastic leukemia (ALL) has several clinical manifestations on the base of bone marrow infiltration and the extent of extra medullary involvement. Anterior chest wall bulging as an initial presentation is rare. The aim of this case report study is to present a 3.5 year-old-boy presented with anterior chest wall bulging without history of trauma since 1 month ago. Tc99m-MDP bone scan showed multifocal active bony pathology in sternum, lower thoracic and all lumbar vertebra and right sacral ala. Bone marrow aspiration and biopsy was in favor of acute precursor lymphoblastic leukemia, B cell type. Therefore, acute lymphoblastic leukemia has different initial presentation and anterior chest wall bulging can be a rare initial sign of acute lymphoblastic leukemia in children.

Effective in Vivo Targeting of BCP-ALL in a NOD/SCID/huALL Mouse Model By CD70 Directed Immunotherapy

Acute lymphoblastic leukemia (ALL) is the most frequent malignant disorder in children and adolescents. Despite successful treatment, relapse of the disease remains a major problem and is associated with poor prognosis. This emphasizes the need for novel treatment strategies to be applied in addition to established chemotherapy regimens without increasing general toxicity.Previously, we described a strong association of leukemia cell engraftment of primary patient B cell precursor (BCP) ALL samples transplanted in a NOD/SCID/huALL mouse model and patient outcome. Rapid onset of leukemia related morbidity (time to leukemia, TTLshort) is indicative for patient relapse and characterized by a specific gene expression profile. Among the top differentially regulated genes, the gene coding for CD70 was identified to be significantly up-regulated in TTLshort/high risk ALL. CD70 is a member of the tumor necrosis factor (TNF) family expressed on activated B- and T-lymphocytes and dendritic cells. Binding of CD70 to its receptor CD27 is involved in regulation of T- and B-cells including priming and generation of memory and plasma cells. CD70 has been described to be constitutively expressed on different cancers including hematological malignancies. However, expression and targeting of CD70 in B- cell precursor lymphoblastic leukemia has so far not been investigated.In this study, we addressed expression of CD70 in patient-derived primograft leukemia samples and primary patient specimens obtained at diagnosis from pediatric patients. Furthermore, we evaluated CD70 as a therapeutic target for directed immunotherapy in vitro and in our BCP-ALL xenograft system in vivo.Flow cytometric analyses of CD70 surface expression in all together 19 patient-derived xenograft samples (TTLshort n= 7, TTLlong n=12) revealed a higher expression of CD70 on ALL cells with a TTLshort/early relapse phenotype compared to TTLlongsamples. We also investigated expression of the CD70 receptor CD27 and found no significant difference in surface expression between both TTL subgroups.Moreover, we investigated the transcript expression levels of 198 BCP-ALL specimens obtained at diagnosis. Interestingly, we found a heterogenous expression of CD70 with no association to cytogenetic subgroups, minimal residual disease (MRD) risk classes or patient outcome. Importantly, a significant higher CD70 expression was found in leukemia samples compared to healthy bone marrow controls indicating a general over-expression in BCP-ALL. CD27 however, did not show different transcript expression including healthy bone marrow controls.To take advantage of increased CD70 expression in BCP-ALL, we addressed CD70 as therapeutic target for immunotherapy. Co-culture in vitro experiments of primograft ALL cells with NK cells in the presence of specific anti-CD70 antibodies revealed five-fold increased antibody-dependent cell-mediated cytotoxicity (ADCC) as compared to the respective isotype control.To evaluate the efficacy of CD70 directed immunotherapy, we assessed leukemia development in NOD/SCID mice upon transplantation of primograft ALL with high CD70 expression either incubated with anti-CD70 antibody or the respective isotype control. Most importantly, a marked reduction of leukemia load in peripheral blood, bone marrow and spleens of the animals was detected in anti-CD70 treated cases. This indicates, that CD70 provides an immunotherapeutic target on ALL cells inducing ADCC by NK cells present in NOD/SCID mice. Most interestingly, this effect could be abrogated both by NK-cell depletion (pre-treatment with anti-mouse CD122 antibodies) in the recipient animals and by using NK-cell deprived NSG mice as recipients, confirming that decreased in vivo leukemia growth upon anti-CD70 treatment is mediated by NK-cell induced cytotoxicity of anti-CD70 bearing CD70 positive ALL cells.Taken together, we identified significantly up-regulated CD70 expression in BCP-ALL with varying expression among molecular and prognostic subgroups. BCP-ALL samples with high surface expression of CD70, as detected by flowcytometry, can be targeted by directed immunotherapy with anti-CD70 antibodies leading to efficient NK-cell dependent lysis of leukemia cells in vitro and decreased growth in an in vivo BCP-ALL model. Thus, CD70 provides a novel target for directed immunotherapy of BCP-ALL. DisclosuresNo relevant conflicts of interest to declare.