Involvement In Acute Lymphoblastic Leukemia Research Articles

The Role of PTEN in Chemoresistance Mediated by the HIF-1α/YY1 Axis in Pediatric Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. Current chemotherapy treatment regimens have improved survival rates to approximately 80%; however, resistance development remains the primary cause of treatment failure, affecting around 20% of cases. Some studies indicate that loss of the phosphatase and tensin homolog (PTEN) leads to deregulation of the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway, increasing the expression of proteins involved in chemoresistance. PTEN loss results in deregulation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and induces hypoxia-inducible factor 1-alpha (HIF-1α) expression in various cancers. Additionally, it triggers upregulation of the Yin Yang 1 (YY1) transcription factor, leading to chemoresistance mediated by glycoprotein p-170 (Gp-170). The aim of this study was to investigate the role of the PTEN/NF-κB axis in YY1 regulation via HIF-1α and its involvement in ALL. A PTEN inhibitor was administered in RS4;11 cells, followed by the evaluation of PTEN, NF-κB, HIF-1α, YY1, and Gp-170 expression, along with chemoresistance assessment. PTEN, HIF-1α, and YY1 expression levels were assessed in the peripheral blood mononuclear cells (PBMC) from pediatric ALL patients. The results reveal that the inhibition of PTEN activity significantly increases the expression of pAkt and NF-κB, which is consistent with the increase in the expression of HIF-1α and YY1 in RS4;11 cells. In turn, this inhibition increases the expression of the glycoprotein Gp-170, affecting doxorubicin accumulation in the cells treated with the inhibitor. Samples from pediatric ALL patients exhibit PTEN expression and higher HIF-1α and YY1 expression compared to controls. PTEN/Akt/NF-κB axis plays a critical role in the regulation of YY1 through HIF-1α, and this mechanism contributes to Gp-170-mediated chemoresistance in pediatric ALL.

JAK2 Kinase Fusion and Myeloid Involvement in Acute Lymphoblastic Leukemia

Introduction JAK2 fusion genes (FGs) are rare but can be found in various phenotypes of myeloid or lymphoid malignancies. Both in the 5th edition of the World Health Organization Classification of Haematolymphoid Tumors (WHO-HAEM5, PMID: 35732831) and the International Consensus Classification (ICC, PMID: 35767897), JAK2-FGs were included in the diagnostic entity of myeloid/lymphoid neoplasms with eosinophilia and tyrosine kinase gene fusions (MLN-TK or MLN-eo), and both suggest MLN-TK/eo as a priority diagnostic entity. It is highlighted in ICC that cases presenting as acute lymphoblastic leukemia (ALL) with M/LN-eo differ from de novo B-ALL and T-ALL in the involvement of myeloid cells. There is still a lack of research reports about how many JAK2-FG-positive ALL cases developed from M/LN-eo or had myeloid involvement. Methods A total of 21 ALL cases (Table 1) were enrolled in this study. The positive for JAK2-FGs were determined by G-banding karyotype analysis, transcriptome sequencing (RNA-seq; PMID: 34135310), reverse transcription PCR (RT-PCR), and interphase fluorescence in situ hybridization (I-FISH) with the JAK2 break-apart probe. Gene mutation analysis (PMID: 29932212) was also performed. According to whether there were FISH-JAK2 split signals in rod and lobed nuclear cells (Figure 1A), the cases were divided into My+ group (with myeloid involved) and My- group (without myeloid involved), respectively. Results Among these cases, there were 14 males and 7 females (M:F = 2:1); 15 B-ALL and 6 T-ALL cases; the median age was 22 years old (range 5-55). All cases were positive for JAK2-FGs in RNA-seq analysis, and all FGs were verified by RT-PCR and Sanger sequencing. A total of 16 JAK2 partner genes were detected (Table 1, Figure 1B/C), of which FNBP1, CDK5RAP2, GTF2I, and C14orf 93 have not been reported previously. All cases retained the complete kinase domain of the JAK2 gene, and 13 cases retained part or all of the JAK2 pseudo-kinase domain in their JAK2-FGs. Karyotype abnormalities were detected in 17 cases (81%), with 9p24 involved in 10 cases (48%), abnormalities other than 9p24 were observed in 7 cases, and karyotype was normal in 4 patients. FISH-JAK2 inspection showed split signals in 20 cases and showed negative results in case #21 who with a complex karyotype abnormality. Twelve cases (12/20=60%) showed JAK2 split signals in rotundum, rod, and lobed nuclei (Table 1, My+ group), suggesting both lymphoid and myeloid lineages were involved. We further investigated samples of 4 cases at the time of morphological complete remission after induction chemotherapy, of which the immunophenotype analysis was also negative for lymphoid blast cells. FISH-JAK2 inspection showed split signals accounting for 13%~79% of the nucleus, with split signals in the rod/lobed nucleus, and RT-PCR also confirmed abundant JAK2-FG transcripts in these samples. There were 8 B-ALL and 4 T-ALL cases in the My+ group, of which 2 T-ALL cases were also positive for STIL::TAL1 fusion, and 3 cases experienced eosinophilia during the course of the disease (Table 1). The 5 cases whose JAK2 partner genes were lymphoid development-related transcription factors (ETV6, PAX5) were all in the My- group. All My- cases and #21 had no documented eosinophilia. Among the 21 cases in this cohort, 13 had white blood cell (WBC) counts >50×10 9/L at diagnosis, and no significant hepatosplenomegaly. The total follow-up time was 2-89m, with a median overall survival (OS) time of 17m. Fourteen cases underwent allo-HSCT, and the median OS was 29.5m in the HSCT group and 9m in the non-HSCT group, respectively. The median OS was 15.5m in the My+ group and 29m in the My- group. The leukemia cells in these cases are prone to be in a high and rapid proliferation state, and they generally have a short OS and dismal prognosis. Conclusions Myeloid involvement accounted for 60% of JAK2-FG positive ALL and My+ cases had a worse prognosis and shorter OS than My- cases in this study. I-FISH inspection can be used as a method to determine whether there was myeloid involvement and provide valuable information for accurate analysis. The incidence of JAK2-FGs is low in ALL, but the fusion partners are diverse. This group of cases is more likely to have cryptic JAK2 translocations and with dismal prognosis. The phenotype of JAK2-FG malignancies is jointly determined by multiple factors under the influence of partner genes, accompanying fusion genes, and accompanying mutations.

Investigating residual leukemic cells in acute lymphoblastic leukemia: a practical approach using a streamlined interphase fluorescence in situ hybridization method on cerebrospinal fluid

IntroductionA precise diagnosis of central nervous system involvement in acute lymphoblastic leukemia (ALL) requires comprehensive knowledge of morphological analysis, with a focus on the quantity and quality of cells being examined. Some research has utilized techniques such as immunocytochemistry, flow cytometry, polymerase chain reaction (PCR), and interphase fluorescence in situ hybridization (iFISH) on cerebrospinal fluid (CSF) cytospin samples to detect any remaining leukemic cells in the CSF. To obtain reliable results using immunocytochemistry and flow cytometry, it is essential to use freshly collected specimens within a limited timeframe. At the same time, PCR requires a sufficient number of cells for DNA extraction. On the other hand, the iFISH procedure on CSF cytospin samples can be challenging and requires practice. Therefore, there is a need for a fast, easy method that will be affordable and marketable in laboratories where the above methods are not available, or the sample is insufficient to use those methods.MethodsThe samples were prepared by centrifugation of 1 mL aliquots of CSF collected into EDTA tubes. The CSF sample was centrifuged at 3000 rpm for 3 min, the supernatant was removed, and the pellet was placed in KCl hypotonic solution for 5 min at 37 °C. Other steps (fixation, hybridization, wash steps, and analysis) were the same as in the standard protocol for blood samples. The BCR-ABL1 rearrangements were performed and evaluated in 200 interphase cells.Results90% of Ph(+) cells were found in CSF.ConclusionWe propose a significantly streamlined iFISH method for detecting blast/residual leukemic cells in acute lymphoblastic leukemia using CSF as a complementary test option.

CXCR4 antagonists disrupt leukaemia-meningeal cell adhesion and attenuate chemoresistance.

The effective prophylaxis and treatment of central nervous system (CNS) involvement in acute lymphoblastic leukaemia (ALL) remains a significant clinical challenge. Developing novel and more effective CNS-directed therapies has been hampered, in part, by our limited understanding of the leukaemia niche in the CNS relative to the bone marrow. Accordingly, defining the molecular and cellular components critical for the establishment and maintenance of the CNS leukaemia niche may lead to new therapeutic opportunities. In prior work we showed that direct intercellular interactions between leukaemia and meningeal cells enhance leukaemia chemoresistance in the CNS. Herein, we show that the CXCR4/CXCL12 chemokine axis contributes to leukaemia-meningeal cell adhesion. Importantly, clinically tested CXCR4 antagonists, which are likely to cross the blood-brain and blood-cerebral spinal fluid barriers and penetrate the CNS, effectively disrupted leukaemia-meningeal cell adhesion. Moreover, by disrupting these intercellular interactions, CXCR4 antagonists attenuated leukaemia chemoresistance in leukaemia-meningeal cell co-culture experiments and enhanced the efficacy of cytarabine in targeting leukaemia cells in the meninges in vivo. This work identifies the CXCR4/CXCL12 axis as an important regulator of intercellular interactions within the CNS leukaemia niche and supports further testing of the therapeutic efficacy of CXCR4 antagonists in overcoming CNS niche-mediated chemoresistance.

Prophylactic Central Nervous System Irradiation Is Not Indispensable in Adult Patients with Acute Lymphoblastic Leukemia: A Multicenter Retrospective Cohort Study

Objective:Studies comparing the efficacy and safety of prophylactic regimens for central nervous system (CNS) involvement in acute lymphoblastic leukemia (ALL) are scarce in adults. This multicenter retrospective study aimed to compare the efficacy of prophylactic regimens with and without CNS irradiation on the development of CNS relapse during follow-up.Materials and Methods:This was a multicenter comparative cohort study. A total of 203 patients were included from four tertiary care centers in Turkey. Patients were divided into two groups according to whether they received CNS irradiation or not. The groups were analyzed retrospectively regarding patient and disease characteristics, with the main focus being CNS relapse.Results:While 105 patients received chemotherapy-based prophylaxis, 98 patients received additional CNS irradiation. These groups were statistically comparable in terms of demographic characteristics and risk factors for CNS involvement. In the irradiation group, patients were younger and had more stem cell transplants. In a median of 23.8 (11.1-62.4) months, there was no difference between the two groups regarding CNS relapse-free survival (log-rank p=0.787).Conclusion:Craniospinal irradiation may not be indispensable for every adult patient with ALL, similarly to pediatric patients. It is crucial to avoid the long-term toxicities of radiation, especially in patients with long life expectancy. Craniospinal irradiation may be reserved for therapeutic use in cases of CNS relapse and prophylaxis for some high-risk patients.

Metabolomic profiling of cerebrospinal fluid reveals an early diagnostic model for central nervous system involvement in acute lymphoblastic leukaemia

The pathogenesis of central nervous system involvement (CNSI) in patients with acute lymphoblastic leukaemia (ALL) remains unclear and a robust biomarker of early diagnosis is missing. An untargeted cerebrospinal fluid (CSF) metabolomics analysis was performed to identify independent risk biomarkers that could diagnose CNSI at the early stage. Thirty-three significantly altered metabolites between ALL patients with and without CNSI were identified, and a CNSI evaluation score (CES) was constructed to predict the risk of CNSI based on three independent risk factors (8-hydroxyguanosine, l-phenylalanine and hypoxanthine). This predictive model could diagnose CNSI with positive prediction values of 95.9% and 85.6% in the training and validation sets respectively. Moreover, CES score increased with the elevated level of central nervous system (CNSI) involvement. In addition, we validated this model by tracking the changes in CES at different stages of CNSI, including before CNSI and during CNSI, and in remission after CNSI. The CES showed good ability to predict the progress of CNSI. Finally, we constructed a nomogram to predict the risk of CNSI in clinical practice, which performed well compared with observed probability. This unique CSF metabolomics study may help us understand the pathogenesis of CNSI, diagnose CNSI at the early stage, and sequentially achieve personalized precision treatment.

Leukemic Optic Neuropathy: A Harbinger of Relapse in Acute Lymphoblastic Leukemia

Background: Leukemic infiltration of the optic nerve is a neuro-oncologic emergency and also a sign of extramedullary central nervous system relapse. It presents a clinical dilemma in the early stages due to multiple differentials. Patients with leukemia receive radiation and chemotherapy are thus, susceptible to inflammatory, toxic, and infectious causes of optic neuropathy, besides infiltration with tumor cells. Clinical Description: A 15-year-old boy treated for acute lymphoblastic leukemia (ALL) and in remission for 7 months, presented with the unilateral decreased vision for 7 days. A structured evaluation was done, comprising visual acuity, color vision, field of vision, fundus, ophthalmoscopy, ultrasound b-scan, and magnetic resonance imaging of the orbit. The final diagnosis was leukemic infiltration of the optic nerve. Cerebrospinal fluid (CSF) analysis confirmed the presence of atypical lymphocytes. Management: The patient was diagnosed with extramedullary relapse of ALL. Since there are no standard guidelines, a literature review was performed, and the treating team decided to start the patient on stand-alone chemotherapy. Symptomatic resolution became apparent within 10 days. On follow-up, the optic nerve lesion resolved with residual gliosis in the surrounding retina. The CSF has become clear, and the patient is now considered to be in remission. Conclusion: It is important to use a structured clinical approach coupled with investigations to recognize the ocular involvement of ALL (especially in younger patients). There is a need for a regular routine ophthalmological examination in patients in remission for early detection of a relapse. There is a strong felt need for pediatric hemato-oncologists to plan research in this area to generate data so that recommendations for the management of extramedullary relapses are formulated.

Functional CRISPR Screening Identifies Ptprg As a Driver of Migration and Adhesion in NSD2-E1099K ALL

Background: Acute Lymphoblastic Leukemia (ALL) is the most common childhood cancer and frequently infiltrates the central nervous system (CNS). CNS-directed therapy is currently limited to intrathecal and systemic high-dose methotrexate, or less commonly craniospinal irradiation, both of which are associated with substantial neurotoxicity. A lack of mechanistic understanding of the mechanisms of CNS infiltration presents an obstacle for the development of more specific and less toxic therapeutic approaches. We previously showed that ALL cells with a specific mutation (E1099K) in the histone methyltransferase NSD2 have aggressive CNS tropism by not only infiltrating the leptomeninges but also the brain parenchyma in murine xenografts models. Analysis of cBioPortal data shows that NSD2-E1099K is associated with a higher rate of testicular involvement in ALL also suggesting more aggressive infiltration behavior of the tumor. Accordingly, using gene editing to revert mutant NSD2 back to wild-type, we also showed that NSD2-E1099K cells have an enhanced ability to migrate and adhere in vitro. RNA-seq data on four NSD2-E1099K cell lines revealed genes that may play a role in ALL brain infiltration. However, it remains unknown which of those upregulated genes could be potential therapeutic targets against CNS leukemia.Aim: This study aims to Identify therapeutically targetable genes that are important for migration of NSD2-E1099K ALL cellsMethods: Using a focused CRISPR-gene-knockout library of 5600 sgRNAs directed against 500 genes upregulated in NSD2-E1099K cells, we ascertained the necessity of the selected genes for migration in the RCH-ACV cell line. Candidate genes were evaluated for cellular dependency using a CRISPR-loss of function screen and the cancer dependency map portal. Overexpression of the candidate genes in NSD2-E1099K cell lines was confirmed with qPCR analysis. Candidate genes were validated by individual shRNA knockdown followed by migration and adhesion assays.Results: Our study identified genes whose knockout led to enhancement of migration and others whose knockout resulted in inhibition of migration. Protein Tyrosine Phosphatase Receptor Type G (PTPRG) was one of the top candidate genes whose knockout resulted in inhibition of migration. Dependency map analysis showed that PTPRG is not a commonly essential gene and a CRISPR-based-loss-of function screen performed in parallel to the migration screen confirmed that ALL cell survival is not dependent on PTPRG. We also found that PTPRG is overexpressed in multiple NSD2-E1099K ALL cell lines. Individual Knockdown of PTPRG in NSD2-E1099K ALL cell lines not only inhibited migration, but also led to a loss of adhesion ability to endothelial cells of the Blood Brain Barrier.Conclusions: Our findings implicate PTPRG as an important modulator of migration and adhesion in ALL cells and a potential therapeutic target for preventing ALL brain infiltration, especially in NSD2-E1099K ALL. DisclosuresLicht: Epizyme: Research Funding.

Applicability and Prognostic Significance of Flow Cytometric Cerebrospinal Fluid Investigation in Children with Acute Lymphoblastic Leukemia

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. DisclosuresNo relevant conflicts of interest to declare.

Testicular involvement of acute lymphoblastic leukemia in children and adolescents: Diagnosis, biology, and management.

Acute lymphoblastic leukemia (ALL) in children and adolescents can involve the testes at diagnosis or upon relapse. The testes were long considered pharmacologic sanctuary sites, presumably because of the blood-testis barrier, which prevents the entry of large-molecular-weight compounds into the seminiferous tubule. Patients with testicular involvement were historically treated with testicular irradiation or orchiectomy. With the advent of contemporary intensive chemotherapy, including high-dose methotrexate, vincristine/glucocorticoid pulses, and cyclophosphamide, testicular leukemia present at diagnosis can be eradicated, with the risk of testicular relapse being 2% or lower. However, the management of testicular leukemia is not well described in the recent literature and remains relevant in low- and middle-income countries where testicular relapse is still experienced. Chemotherapy can effectively treat late, isolated testicular B-cell ALL relapses without the need for irradiation or orchiectomy in patients with an early response and thereby preserve testicular function. For refractory or early-relapse testicular leukemia, newer treatment approaches such as chimeric antigen receptor-modified T (CAR-T) cell therapy are under investigation. The control of testicular relapse with CAR-T cells and their penetration of the blood-testis barrier have been reported. The outcome of pediatric ALL has been improved remarkably by controlling the disease in the bone marrow, central nervous system, and testes, and such success should be extended globally. LAY SUMMARY: Acute lymphoblastic leukemia (ALL) in children and adolescents can involve the testes at diagnosis or upon relapse. Modern intensive chemotherapy has largely eradicated testicular relapse in high-income countries. Consequently, most current clinicians are not familiar with how to manage it if it does occur, and testicular relapse continues to be a significant problem in low- and middle-income countries that have not had access to modern intensive chemotherapy. The authors review the historical progress made in eradicating testicular ALL and use the lessons learned to make recommendations for treatment.

Acute Lymphoblastic Leukemia with Primary Testicular Involvement in a Pre-pubertal Male: A Case Report

Acute lymphoblastic leukaemia is the most common malignancy in paediatric patients; its diagnosis is usually easy to establish as malignant lymphoblasts invade the bone marrow and peripheral blood. Patients with Acute lymphoblastic leukaemia (ALL) may initially present with pancytopenia and a hypoplastic or hyperplastic bone marrow. Fever is common at presentation, and despite neutropenia, sepsis is rarely seen. Other common clinical manifestations include fatigue, pallor, petechiae, bleeding, lymphadenopathy and hepatosplenomegaly. Presentation with primarily testicular disease is exceedingly rare in acute lymphoblastic leukemia. The unusual presentation of a primary left testicular involvement in ALL is being highlighted in this case report. We present an 11 year old boy who presented with fever, generalized body pain, abdominal discomfort as well as left testicular swelling. Bone marrow studies, Full blood count and peripheral blood film appearance were typical of acute lymphoblastic leukaemia. Ultrasound findings showed left scrotal enlargement with reduced and coarse parenchymal echotexture, while cytogenetic studies revealed positive Philadelphia chromosomes. There was good response to standard ALL therapy. Clinical remission with normal left testicular size was noted at the end of induction phase of therapy.

Socioeconomic Status Is Associated with Prognostic Factors in Childhood Acute Lymphoblastic Leukemia - Implications for Outcomes: A Report from the Children's Oncology Group

Introduction: Acute lymphoblastic leukemia (ALL) is the most common form of childhood cancer. Historical studies have found an association between ALL incidence with higher socioeconomic status (SES), though newer findings are contradictory for this association. Nevertheless, it has been well established that inferior outcomes in pediatric ALL are associated with lower SES. We sought to identify if specific ALL disease characteristics at diagnosis are associated with SES to determine if underlying biology plays a role in the known association between inferior outcomes and lower SES. Methods: Demographic and disease characteristics from children and young adults (aged 2-30 years) diagnosed with ALL 2004-2017 and treated on Children's Oncology Group (COG) frontline treatment protocols (N=4,726, AALL17D2) were matched 1:1 (by age, sex, race, and sampling year) and compared to National Health and Nutrition Examination Survey (NHANES) controls. SES for cases was defined using census data to determine the percent of population in a ZIP code living at or below the federal poverty threshold. SES for controls was defined by the individual's poverty income ratio. Chi-square testing and multivariate logistic regressions were performed, adjusting for sex, race/ethnicity, and age, to assess associations between SES and ALL, as well as specific risk-stratifying ALL disease characteristics, including cytogenetics and central nervous disease (CNS) involvement. Results: ALL patients (72% B-ALL, 28% T-ALL) were more likely to be male (62%), 58% were non-Hispanic white, 9% non-Hispanic black and 24% identified as Hispanic. By chi-square analysis, a difference in SES was identified between cases and controls (p&lt;0.0001). This was further characterized by multivariate logistic regression, which found a positive significant association between ALL incidence and high SES, when using low SES as the reference group (OR 2.54, 95% CI 2.23-2.89, p &lt; 0.0001). When disease characteristics were evaluated, there was an association between SES and CNS status, with presence of ALL involvement in the CNS (CNS 2 or 3 status) more strongly associated with low SES (p=0.0009). When stratified by sex and race, an association between low SES and CNS status at diagnosis remained among non-Hispanic white males and Hispanic males (p=0.008 and p=0.01, respectively). There was also an association between high SES and the presence of the favorable cytogenetic characteristic trisomy of chromosomes 4 and 10 (p=0.01), though this was not seen in a specific group when stratified by sex and race. Among non-Hispanic white males, an association was found between low SES and presence of rearrangement of the MLL gene, a poor prognostic cytogenetic characteristic in ALL (p=0.04). Among Hispanic males, an association was found between high SES and the presence of an ETV6-RUNX1 translocation status, a favorable prognostic cytogenetic characteristic in ALL (p=0.05). These associations were not seen when all cases were assessed together. We did not find an association between SES and immunophenotype, hypodiploidy, initial white blood cell count, or BCR-ABL1 fusion status Conclusions: To our knowledge, this is the first study to assess associations between SES and ALL disease characteristics that have known prognostic implications in disease outcome. We found that poor prognostic features of ALL including CNS involvement at diagnosis (regardless of sex and race) and presence of MLL rearrangement specifically in non-Hispanic white males, were both associated with low SES. In contrast, favorable prognostic features of ALL were associated with high SES, such as the presence of trisomy of chromosomes 4 and 10, and among Hispanic males the presence of an ETV6-RUNX1 translocation. Our study also replicated the association between ALL diagnosis and high SES, which has been a controversial finding. Nonetheless, our findings suggest that there may be biological factors that play a role in the known association between inferior ALL outcomes and low SES. Further studies to better elucidate this potential biologic role are needed as there may be important implications for future treatment options that could improve outcomes for children with ALL who have low SES, as well as improve disparate outcomes currently seen in ALL treatment. Disclosures No relevant conflicts of interest to declare.

Absolute count of leukemic blasts in cerebrospinal fluid as detected by flow cytometry is a relevant prognostic factor in children with acute lymphoblastic leukemia.

Usually, central nervous system (CNS) involvement in acute lymphoblastic leukemia (ALL) is diagnosed by cytomorphology (CM) of cerebrospinal fluid (CSF) on cytospin slides. Multicolor flow cytometry (MFC) provides the opportunity to detect low numbers of leukemia cells undetectable by CM. The present study aimed at evaluating the clinical significance of MFC for the diagnosis of CNS involvement at initial manifestation of childhood ALL. In 155 children with ALL, CSF samples were studied in parallel by CM and MFC. Patients were treated according to protocol ALL-MB-2008 for childhood ALL. The prognostic impact of the leukemia burden in CSF was determined categorizing the findings as positive/negative. In addition, the absolute blast cell count per 1ml of CSF was studied as a continuous variable. CSF positivity was significantly more frequent using MFC compared with CM (35.3% vs. 15.3% of patients). The outcome of MFC-positive and MFC-negative patients was not different in clinically relevant patient risk groups-CNS1, standard and intermediate-risk groups. Using the quantitative approach, at the threshold level of 20 blasts per ml of CSF, patients could be divided into two groups with a significantly different outcome, irrespective of the clinical risk group, the type of CNS-directed therapy, and the CNS status determined by CM. Our data do not support the concept of re-stratification and modification of therapy based on qualitative CSF investigation by MFC. However, MFC is a highly sensitive technique of CSF investigation improving the definition of CNS involvement in childhood ALL, and quantitative measurement of blast cells in CSF, if well-organized, can be a useful additional tool for stratification of patients in clinical trials.

Central nervous system involvement in acute lymphoblastic leukemia

It is well known that acute lymphoblastic leukemia (ALL) cells can invade the central nervous system (CNS), but the underlying mechanism of such invasion is still unclear. We discovered the direct routes taken by ALL cells when migrating into CNS in ALL model mice. We observed that ALL cells migrate along the external surface of vessels that pass directly between the vertebral or calvarial bone marrow and the subarachnoid space. The basement membrane of these bridging vessels is enriched in laminin. The laminin is recognized by integrin α6, which is expressed by ALL cells. The interaction between integrin α6 and laminin mediated the invasion of ALL cells. Furthermore, the expression of integrin α6 depends on PI3Kδ activity. Mice with ALL xenografts were treated with a PI3Kδ inhibitor, which decreased integrin α6 expression on ALL cells. This resulted in significant reductions in blast counts in the cerebrospinal fluid and in CNS disease symptoms. Our data suggest that the PI3Kδ inhibitor has potential to prevent CNS involvement in ALL.

Acute leukemia after cytotoxic treatment in a child with nephrotic syndrome

Renal involvement in acute lymphoblastic leukemia (ALL) occurs due to several factors including leukemic infiltration of the kidneys, therapy-related side effects such as tumor lysis syndrome, nephrotoxic drugs, and septicemias. A 3-year-old boy with nephrotic syndrome (NS) who was previously treated with prednisolone and cyclosporine A for 14 months after the initial diagnosis of NS, presented to the emergency department with fever, breathing difficulty, generalized edema, and body pain with pallor, without evidence of lymphadenopathy, hepatosplenomegaly, petechiae, or purpura. On investigation, peripheral blood smear showed blast cells &gt;80% and bone marrow aspiration showed complete replacement of the marrow with L1 lymphoblasts, consistent with a diagnosis of ALL. The exact mechanism of developing acute leukemia after cytotoxic treatment has not been established; the possibility must be considered that the incidence of this malignant disease is increased after cytotoxic treatment for nonmalignant diseases.

Use of the Polymerase Chain Reaction as a Complementary Method for the Detection of Central Nervous System Involvement in Children and Adolescents with Acute Lymphoblastic Leukemia.

Cytological analysis of the cerebrospinal fluid (CSF) remains the most widely used method for diagnosing central nervous system (CNS) involvement in acute lymphoblastic leukemia (ALL). This study aimed at evaluating the use of polymerase chain reaction (PCR), in comparison to other methods, for the assessment of the presence of blast cells in the CSF at the time of diagnosis of ALL. This was a prospective, single-centre, study enrolling all patients up to the age of 18 years who were admitted to a university hospital between November 2011 and November 2014 with a diagnosis of ALL and from whom it was possible to draw a sufficient amount of CSF for analysis by conventional cytology (CT), immunophenotyping (IMP), and PCR. A total of 46 CSF samples from 44 ALL pediatric patients were included. CT was performed in all samples, IMP in 44, and PCR in 34. Thirteen (28.2%) samples showed positive results: two by CT, four by IMP, four by PCR, and three by both IMP and PCR. The results of this study showed that PCR should be considered a complementary method for the evaluation of the CSF in ALL patients at diagnosis.

Outcome Analysis of Pediatric Patients with Acute Lymphoblastic Leukemia Treated with Total Body Irradiation–Free Allogeneic Hematopoietic Stem Cell Transplantation: Comparison of Patients with and Without Central Nervous System Involvement

Hematopoietic stem cell transplantation (HSCT) with a non–total body irradiation (TBI) conditioning regimen has proven feasible for treating patients with acute lymphoblastic leukemia (ALL). However, it is commonly believed that for extramedullary involvement of ALL in sanctuary sites, such as the central nervous system (CNS), TBI shall not be abandoned. In this study, the outcomes of pediatric ALL patients with CNS involvement (CNS+) and without CNS involvement (CNS−) treated with TBI-free allogeneic HSCT were retrospectively compared. The patients received a TBI-free busulfan plus cyclophosphamide conditioning regimen. Comparing CNS+ (n = 27) and CNS− (n = 134) patients, the 5-year probabilities of relapse (44.4% versus 41.8%; P = .799), disease-free survival (DFS; 48.1% versus 43.3%; P = .642) and overall survival (OS; 51.9% versus 47.0%; P = .646) were not significantly different. Although transplantation-related mortality (TRM) was higher in the CNS− patients, the difference between the 2 groups was not significant (3.7% versus 12.7%; P = .177). In multivariate analysis, there were no significant between-group differences in OS (P = .502), DFS (P = .424), relapse rate (P = .226), or TRM (P = .117). These findings suggest that HSCT using a non–TBI-containing conditioning regimen can lead to similar outcomes in pediatric ALL patients with and without CNS involvement. TBI-free allogeneic HSCT might be feasible and effective for CNS+ ALL patients.

Central nervous system involvement in acute lymphoblastic leukemia is mediated by vascular endothelial growth factor

AbstractIn acute lymphoblastic leukemia (ALL), central nervous system (CNS) involvement is a major clinical concern. Despite nondetectable CNS leukemia in many cases, prophylactic CNS-directed conventional intrathecal chemotherapy is required for relapse-free survival, indicating subclinical CNS manifestation in most patients. However, CNS-directed therapy is associated with long-term sequelae, including neurocognitive deficits and secondary neoplasms. Therefore, molecular mechanisms and pathways mediating leukemia-cell entry into the CNS need to be understood to identify targets for prophylactic and therapeutic interventions and develop alternative CNS-directed treatment strategies. In this study, we analyzed leukemia-cell entry into the CNS using a primograft ALL mouse model. We found that primary ALL cells transplanted onto nonobese diabetic/severe combined immunodeficiency mice faithfully recapitulated clinical and pathological features of meningeal infiltration seen in patients with ALL. ALL cells that had entered the CNS and were infiltrating the meninges were characterized by high expression of vascular endothelial growth factor A (VEGF). Although cellular viability, growth, proliferation, and survival of ALL cells were found to be independent of VEGF, transendothelial migration through CNS microvascular endothelial cells was regulated by VEGF. The importance of VEGF produced by ALL cells in mediating leukemia-cell entry into the CNS and leptomeningeal infiltration was further demonstrated by specific reduction of CNS leukemia on in vivo VEGF capture by the anti-VEGF antibody bevacizumab. Thus, we identified a mechanism of ALL-cell entry into the CNS, which by targeting VEGF signaling may serve as a novel strategy to control CNS leukemia in patients, replacing conventional CNS-toxic treatment.

Higher rate of central nervous system involvement by flow cytometry than morphology in acute lymphoblastic leukemia.

Central nervous system (CNS) involvement in acute lymphoblastic leukemia (ALL) is diagnosed traditionally by cytopathology (CP) of the cerebrospinal fluid (CSF). Role of flow cytometry (FC) to diagnose CNS involvement has not been extensively investigated. We aimed to detect CNS involvement in 42 ALL patients (33 B-ALL, nine T-ALL) at diagnosis by FC and comparing it with CP and to correlate it with known risk factors for CNS disease like Lactate dehydrogenase (LDH). A receiver operating characteristic curve was used to determine the cutoff of LDH to predict CSF involvement. For the analysis of categorical/quantitative variables, Fisher's exact test was used. For the analysis of continuous variables, Mann-Whitney test was used. A P value of <.05 was taken as significant. CP and FC were positive in five (11.9%) and 11 patients (26.14%) respectively with FC detecting a significantly higher level of involvement (P=.001). All CP-positive cases were FC positive. A LDH value of >472U/L had a sensitivity of 61% and specificity of 62.5% for diagnosis of CSF involvement by FC. CSF FC detects CNS disease in ALL patients at diagnosis at a rate double than CP alone and is statistically associated with an elevated LDH level. It should be incorporated in the evaluation of CSF to detect CNS involvement.

Effect of chimeric antigen receptor-modified T (CAR-T) cells on responses in children with non-CNS extramedullary relapse of CD19+ acute lymphoblastic leukemia (ALL).

10507 Background: Anti-CD19 CAR-T cell therapies have shown high efficacy in inducing durable marrow responses in patients with relapsed/refractory CD19+ ALL. We now report on outcome of 10 patients with extramedullary (EM) involvement of ALL treated with CAR-T, including 5 patients who had EM disease at time of infusion. Methods: We identified patients treated on pediatric phase 1/2a trials of murine (CTL019) or humanized (CTL119) anti-CD19 CAR-T cells for isolated EM or BM/EM relapse of ALL. EM relapse was defined as involvement of non-CNS site by imaging +/- pathology within 12 months (mos) of infusion. Post infusion, patients had diagnostic imaging done at 1, 3, 6, 9, and 12 mos. Results: Among 97 patients receiving CAR-T, ten (CTL019, n=6; CTL119, n=4) were identified who had EM involvement on average 2.3 mos (range 0-9 mos) prior to infusion; including 5/10 at time of infusion. Sites of EM relapses included testes, sinus, parotid, bone, uterus, kidney and skin, and 5 patients had multiple sites of EM involvement. Patients ranged from 2-4 relapses of their ALL pre-CAR-T. Two had isolated EM relapse (sites were parotid and multifocal bony lesions in one; testis and sinus in second). All 10 patients had undergone hematopoietic stem cell transplantation prior to EM relapse, 2 had received radiation directed to the EM site prior to CAR-T. Five patients evaluated by serial imaging had objective responses: 2 had resolution of EM disease by day 28; 2 had resolution by 3 mos; 1 had continued decrease in size of uterine mass at 3 and 6 mos and underwent hysterectomy at 8 mos with no evidence of disease on pathology. In the 4 patients with prior history of skin or testicular involvement, there was no evidence by exam at day 28. One patient had progressive EM disease within 2 weeks of CAR-T cell infusion and died at 6 weeks. Three relapsed with CD19+ disease [1 skin/medullary- died at 38 mos post CAR-T; 2 medullary (1 died at 17 mos, 1 alive at 28 mos)]. The remaining 6 are alive and well at median follow-up of 10 mos (range 3-16 mos) without recurrence of disease. Conclusions: Single agent CAR-T immunotherapy can induce potent and durable responses in patients with EM relapse of their ALL. Clinical trial information: NCT01626495, NCT02374333.