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Analysis of Normal Hematopoietic Stem and Progenitor Cell Contents in Childhood Acute Leukemia Bone Marrow
Archives of Medical Research 47 (2016) 629e643
ORIGINAL ARTICLE
Analysis of Normal Hematopoietic Stem and Progenitor Cell Contents in Childhood Acute Leukemia Bone Marrow Juan Carlos Balandran,a,b,* Eduardo Vadillo,a,b,* David Dozal,c,* Alfonso Reyes-L opez,d c c Antonio Sandoval-Cabrera, Merle Denisse Laffont-Ortiz, Jessica L. Prieto-Chavez,e Armando Vilchis-Ordo~ nez,d Henry Quintela-Nu~ nez del Prado,f Hector Mayani,a g Juan Carlos N u~ nez-Enrıquez, Juan Manuel Mejıa-Arangure,g Briceida L opez-Martınez,d Elva Jimenez-Hernandez,h and Rosana Pelayoa a
Oncology Research Unit, Oncology Hospital, Instituto Mexicano del Seguro Social, Mexico City, Mexico b Departamento de Biomedicina Molecular, CINVESTAV, Mexico City, Mexico c Hospital para el Ni~no, Instituto Materno Infantil del Estado de Mexico, Mexico d Hospital Infantil de Mexico ‘‘Federico Gomez’’, Mexico City, Mexico e Unidad de Investigacion Medica en, Hospital de Especialidades, Instituto Mexicano del Seguro Social, Mexico City, Mexico f Unidad Medica de Alta Especialidad, ‘‘Dr. Victorio de la Fuente Narvaez’’, Instituto Mexicano del Seguro Social, Mexico City, Mexico g Unidad de Epidemiologıa de Investigacion Clınica Hospital de Pediatrıa, Instituto Mexicano del Seguro Social, Mexico City, Mexico h Hospital Pediatrico Moctezuma, Mexico City, Mexico Received for publication October 24, 2016; accepted November 23, 2016 (ARCMED-D-16-00651).
Background and Aims. Childhood acute leukemias (AL) are characterized by the excessive production of malignant precursor cells at the expense of effective blood cell development. The dominance of leukemic cells over normal progenitors may result in either direct suppression of functional hematopoiesis or remodeling of microenvironmental niches, contributing to BM failure and AL-associated mortality. We undertook this study to investigate the contents and functional activity of hematopoietic stem/progenitor cells (HSPC) and their relationship to immune cell production and risk status in AL pediatric patients. Methods. Multiparametric flow cytometry of BM aspirates was performed to classify AL on the basis of lineage and differentiation stages and to analyze HSPC and immune cell frequencies. Controlled co-culture systems were conducted to evaluate functional lineage potentials of primitive cells. Statistical correlations and inter-group significant differences were established. Results. Among 113 AL BM aspirates, 26.5% corresponded to ProB, 19.5% to PreB and 32% contain ProB and PreB differentiation stages, whereas nearly 9% of the cases were T- and 13% myeloid-lineage leukemias. We identified ProB-ALL as the subtype endowed with the highest relative contents of HSPC, whereas T-ALL and PreB-ALL showed a critically reduced size of both HSC and MLP compartments. Notably, lower cell frequencies of HSPC in ProB-ALL correlated to high-risk prognosis at disease debut. Conclusions. HSPC abundance at initial diagnosis may aid to predict the clinical course of ALL and to identify high-risk patients. A clearer understanding of their population dynamics and functional properties in the leukemia setting will potentially pave the way for targeted therapies. Ó 2016 IMSS. Published by Elsevier Inc. Key Words: Acute lymphoblastic leukemia, Stem/progenitor cells, Lymphoid immune cells, Bone marrow, ProB-ALL high risk.
*
These authors contributed equally to this work. Address reprint requests to: Dr. Rosana Pelayo, Oncology Research Unit, Oncology Hospital, IMSS, Av. Cuauhtemoc 330, Colonia Doctores,
06720 Mexico City, Mexico; Phone: (þ52) (55) 5627-6915 ext. 22705 or 22710; FAX: NEEDED; E-mail: [email protected].
0188-4409/$ - see front matter. Copyright Ó 2016 IMSS. Published by Elsevier Inc. http://dx.doi.org/10.1016/j.arcmed.2016.12.004
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Introduction The most important disease endangering the hematopoietic system in pediatric patients is acute leukemia (AL), which constitutes the major cause of death worldwide (1,2). Particularly, childhood AL in Mexico shows among the highest incidence rates with extraordinary heterogeneity and inferior outcomes (3). Almost 85% of the cases compromise the lymphoid linage, whereas 15e20% are from myeloid origins (2). Historically, T-lineage leukemias have been considered the subtype endowed with the most adverse scores (4). However, accumulating evidence of cytopenias and impaired hematopoiesis in some B- and myeloid-AL, often resulting in bone marrow (BM) failure and fatal outcomes has suggested that ineffective hematopoiesis is the clinical hallmark of all AL (4). The high proliferative activity and oligoclonal dominance of leukemic cells over the normal hematopoietic stem and progenitor cells (HSPC) displace them in a dynamic competition for the BM niches (1). As leukemic-initiating cells (LICs) coexist with normal primitive cells within the CD34þ cell compartment, no surface markers can be used so far to distinguish normal HSPC from LICs, making the normal vs. malignant population dynamics still uncertain (5). Hematopoietic primitive cells fulfill their functional production of blood cells in the context of microenvironmental regulation. Emerging research sustains the notion that neoplastic cells are also dependent on the surrounding microenvironment and point to the influence of specialized niches for leukemia progression (6e9). Hematopoietic suppression as a result of excessive leukemic growth may be contributed by direct inhibition of developing cells by soluble factors, by the outcompetition for stem cell niches and by leukemic cell-induced microenvironment remodeling, creating a leukemic sanctuary unsuitable for normal hematopoiesis (10). The biological and molecular mechanisms involved in the displacement of normal hematopoiesis are currently under investigation by a number of groups. Our recent findings suggest that primary B-lineage ALL cells are capable of producing pro-inflammatory cytokines and abnormal amounts of growth factors that interfere with regular hematopoietic proliferation and differentiation processes (11). Moreover, exhaustion of the HSPC compartment has been demonstrated in chronic myeloid leukemia (CML) in a leukemic cell-derived IL-6 dependent manner (12). Elegant animal models have been useful to confirm that leukemic cells hijack and remodel normal mouse niches displacing normal hematopoiesis to favor leukemic progression concomitant to down-regulation of the primary chemokine CXCL12 in BM (13,14). Accordingly, we have reported that cell frequencies of functional HSPC are critically reduced in B-ALL patients (15), with our recent data suggesting that mesenchymal reticular niche within the leukemic BM is not able to sustain ex vivo
normal lymphopoiesis due to a diminished expression of CXCL12, but facilitating instead the maintenance and progression of primary ALL cells (Balandran JC et al., submitted). Of note, abnormal BM mesenchymal stromal cell (MSC) niches in a T-ALL mice model were shown to be unable to support HSPC due to their accelerated senescence (16). Strikingly, minimal residual disease (MRD) detection has been the most important prognostic indicator of treatment success or failure, with the supposed concomitant emergence of normal hematopoiesis responsible for repopulating the BM following remission (17). To date, no reports monitoring HSPC numbers and function in childhood AL or correlation studies with clinical outcomes are recorded. Rebounding of normal hematopoiesis should be reflected in blood and BM cell frequencies close to homeostatic values. Interestingly, experimental data from a competitive repopulation in a pre-clinical model showed a good correlation of HSPC with good prognosis, with increasing doses of normal HSPC delaying the LIC establishment (18). Similar observations have resulted from experimental models of AML (19). Mature B, T, NK and dendritic cells as well as macrophages are a substantial part of the BM microenvironment. As all immune cells derive from HSPC, the evaluation of alterations in such a primitive compartment is of high relevance. In solid tumor settings, immune cell contents constitute a prognosis factor where global outcomes depend on immunoregulatory cell subsets abundance. Further immunity evaluation in cancer will provide better information with prognostic value (20). A novel immunoscore allows classification of malignancies within an immune context where, remarkably, infiltration of subsets of T cells has been associated with good prognosis in several solid tumors (21), whereas activation of T cells in leukemias contribute to tumor surveillance and control (22). In this work the contents and functional activity of HSPC and their relationship to immune cell production and risk status in acute leukemia have been investigated. Among five subtypes of AL (ProB-ALL, PreB-ALL, ProB/PreB-ALL, T-ALL and AML), we found that ProBALL has the highest contents of HSPC, whereas T-ALL and PreB-ALL showed scarce HSC and MLP cells. Notably, lower cell frequencies of HSPC in ProB-ALL correlated to high-risk prognosis. To the best of our knowledge, this is the first report describing BM normal hematopoietic content in the immune context in childhood AL.
Materials and Methods Patient Characteristics and Sample Collection One hundred thirteen children diagnosed with ALL were included in this study: 63 were referred to the ‘‘Hospital para
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Table 1. Patient characteristics Patient 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Age (years) 9 11 9 10 9 8 10 3 14 16 11 15 8 9 14 14 12 8 3 2 9 4 9 9 8 11 10 9 10 15 13 7 8 7 14 7 8 10 3 15 14 5 10 7 12 8 3 4 4 7 11 6 14 14 9 9 5 9 8 9
Sex
Phenotype
M F M M F M M F M M M M M M M M M M F F M M M F F M M F M M F F M M M F F M F F F F F M F F F F F M F F F M F F F F F F
AML AML AML AML AML AML AML AML AML AML AML AML AML AML AML PreB PreB PreB PreB PreB PreB PreB PreB PreB PreB PreB PreB PreB PreB PreB PreB PreB PreB PreB PreB PreB PreB ProB ProB ProB ProB ProB ProB ProB ProB ProB ProB ProB ProB ProB ProB ProB ProB ProB ProB ProB ProB ProB ProB ProB
WBC 3103/mm3
BM Blast (%)
Chromosomal aberrations
Risk
10.5 32 52 45.6 134 129.1 62.8 39.6 59.3 18.5 52.5 45 2.2 187 5.1 293.4 7.1 281 123 188 23 143 121 109 71.7 45.6 112 108.6 110.6 110.8 12.5 104.9 58.3 108.3 53.5 164.5 107.9 4.9 5.1 118.9 58.6 47.9 285.5 34 45.6 78.5 121.4 112 60.4 83.51 -
5 74.9 75 23.6 87 -
Neg t (15,17)(q24;q21) Neg t (15,17)(q24;q21) PML ex3-RARA ex5 t (15,17)(q22;q21) PML ex3-RARA ex5 Neg Neg t (9,22)(q34;q11) BCR ex13 - ABL1 ex2 (b2a2) € ı PBX ex3 t (1,19)(q23;p13) TCF3 ex16, A Neg Neg t (15,17)(q22;q21) PML ex3-RARA ex5 t (12,21)(p13;q22) ETV6 ex5-RUNX1 ex4: t (9,22)(q34;q11) Neg t (1,19)(q23;p13)TCF3-PBX1(PRL) Neg Neg t (12,21)(p13;q22) ETV6 ex5-RUNX1 ex3 Neg Neg Neg Neg -
High High High High High High High High High High High High High High High High High High High High High High High High High High High High High High Standard High Standard Standard High High High High High Standard High High High Standard High High High High Standard High High Standard High High High High Standard High High High
45 62 70.3 65.6 97 0.07 0.07 92.33 70 94.6 67 89 73.5 48.3 67 56 78 67 90.4 98.6 78.6 94.6 56.4 84 0.03 82.53 74 67 87.8 85 80.6 72 70 92.3 83.1 90.8 90 12 65 80.02 78 56 49.5 83.51 -
(continued on next page)
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Table 1 (continued ) Patient 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113
Age (years) 16 5 15 7 8 7 13 8 7 14 9 11 3 7 2 4 3 2 8m 12 3 9 16 4 3 1 3 7 5 6 6 15 5 4 6 2 5 6 14 7 2 2 1 3 13 12 5 15 14 15 3 2 5
Sex
Phenotype
WBC 3103/mm3
BM Blast (%)
Chromosomal aberrations
Risk
M F F M M M F F F M M M M F M F F F M F F M M M F F M M M F M F M M F F F F M M F F M M F M F F F F M M F
ProB ProB ProB ProB ProB ProB ProB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB T-ALL T-ALL T-ALL T-ALL T-ALL T-ALL T-ALL T-ALL T-ALL T-ALL
45.7 34.5 29 89.4 60.3 48.6 117 62.3 100.7 270 119.2 33.9 160.5 37.5 176.3 45.6 125.3 25.6 150 55 9.5 64.6 123 16.5 162.5 45.3 12.4 27.6 26.9 29.4 145.2 43.2 134.5 40.6 15.6 23.5 45.2 59.9 42.1 135.6 791 116 316.2 6.4 79.4 107.3 112.1 231.5
28.5 86.4 83.74 70 81.01 80 71.23 88.5 81 85.9 83 92.2 86.35 87 76.89 90.6 93.88 62.4 93.9 86.6 74.9 89.7 66 70 95 65.4 37.8 68.4 78.6 76.1 67.8 25 89.4 71.37 88.05 76.4 22.8 91.47 82.24 67.3 96.06 86 88 30 87 94.1 89.5 76.4
Neg Neg t (15,17)(q24;q21) Neg t (1,19)(q23;p13)TCF3-PBX1(PRL) Neg Neg t (1,19)(q23;p13) t (12,21)(p13;q22) ETV6 ex5-RUNX1 ex4: Neg Neg Neg t (1,19)(q23;p13)TCF3-PBX1(PRL) Neg t (12,21)(p13;q22) ETV6 ex5-RUNX1 ex4: € ı ERG ex13 t (16,21)(p11;q22) FUS ex7, A t (12,21)(p13;q22) ETV6 ex5-RUNX1 ex4: Neg Neg Neg Neg t (12,21)(p13;q22) Neg Del TAL1 Neg Neg -
High Standard High High High High High High High High High High High High High High High High High High High High High High High High High Standard Standard Standard Standard High Standard High Standard High Standard Standard High High High High High High High High High High High High High High High
el Ni~ no’’, 43 to the ‘‘Hospital Pediatrico Moctezuma’’ and seven to the ‘‘Hospital Infantil de Mexico Federico Gomez’’. Among them, 84.0% were stratified for high-risk disease, whereas 15.9% were stratified for standard-risk according to the blast cell count, age, T-linage and/or genetics criteria; 49.5% of the patients were male and 50.4% were female.
Median age values were 8 years (8 monthse16 years) and 3 years (1e7 years) for the high-risk and standard-risk group, respectively (Table 1). Control samples were obtained from healthy children undergoing minor orthopedic surgery BM specimens were collected by aspiration before any treatment, respecting international and institutional guidelines.
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All procedures were approved by the Ethics, Research and Biosafety Committee of the Hospitals and by the National Committee of Scientific Research at the Mexican Institute for Social Security (Registries CIEICE-007-01-13, R-20123602-29 and R-2015-785-120). All samples were collected after written informed consent from parents.
Cells were classified into phases of cell cycle by using the Cell Cycle Flow Cytometry Analysis Software (FlowJo, Tree Star, Inc.), which calculates the cell frequencies in G1/G0, S and G2/M.
Immunophenotyping by Flow Cytometry
Data were analyzed using STATA v.10.0 (Stata Corporation) and Prism V3.02 (GraphPad) softwares. Differences within groups were established by non-parametric test, considering significant probability values ! 0.05.
White blood cell numbers in BM samples were determined using Turk’s solution and 5 105 leukocytes were added per tube. Cells were stained with hCD45-PE-Cy5, hCD34-APC, hCD19FITC and hCD10-PE (Tube 1). The same cell number was stained with hCD7-APC, hCD56-FITC and hCD13-PE (Tube 2) or with hCD3-PE and hCD14-FITC (Tube 3) (Supplementary Figure 1). In addition, 1 106 leukocytes were stained with a lineage antibody cocktail (Lin: hCD3-PE, hCD8PE, hCD11c-PE, hCD14-PE, hCD19-PE, hCD56-PE and hCD235a-PE), hCD34-APC, hCD38-FITC and hCD45RAPE-Cy5. All antibodies were from BioLegend, San Diego, CA. Red blood cells were lysed with BD pharm lyse (BD Biosciences, San Jose, CA) following antibody staining. Samples were acquired on a CyAn (Beckman Coulter, Pasadena, CA) or a BD FACSCanto II cytometers. Analysis of flow cytometry data was performed using the FlowJo 10.0.8 software. Isolation of HSPC Mononuclear cells (MNC) from BM samples were prepared by using Ficoll-Paque Plus (GE Healthcare Bioscience, Uppsala, Sweden) and CD34þ pool was enriched from MNC with the human CD34 progenitor cell isolation kit (Miltenyi Biotec, San Diego, CA) according to the manufacturer’s instructions. CD34þ fraction was stained with anti-CD34 and anti-lineage antibodies followed by HSPC sorting on the basis of CD34 expression and lacking of linage markers (CD34þLin‒), using a BD FACSAria sorter. Samples were individually manipulated and no sample pooling was performed for any of the conducted experiments. Co-cultures in Lymphoid Conditions HSPC were co-cultured with MS-5 stromal cells for 30 day in triplicate in a-MEM (Gibco, Anaheim, CA) culture medium supplemented with 10% fetal bovine serum (Gibco), 1 ng/mL Flt3-L, 2 ng/mL SCF, 5 ng/mL IL-7, and 10 ng/ mL IL-15 (PeproTech, Rocky Hill, NJ) and contained 100 U/mL penicillin and 100 U/mL streptomycin (Gibco). Cocultures were incubated at 37 C in a humidified atmosphere of 5% of CO2. At the end of the cultures, cells were harvested and viability was also determined. Cell Cycle Analysis After membrane staining with anti-hCD34 and a lineage antibody cocktail, cells were fixed and permeabilized prior to 7-aminoactinomycin (7-ADD) staining (BD Biosciences).
Statistics
Research on Human Subjects As stated, all procedures were approved by the Ethics, Research and Biosafety Committee of the Hospitals and by the National Committee of Scientific Research at the Mexican Institute for Social Security (Registries CIEICE-007-01-13, R-2012-3602-29 and R-2015-785-120). All samples were collected after written informed consent from the parents.
Results Multiparametric Flow Cytometry Immunophenotyping and Classification of Childhood Acute Leukemias We designed a panel based on the lymphoid differentiation surface markers (CD45, CD34, CD10, CD19 and CD7), additional immune lymphoid cells (CD56 and CD3) and myeloid markers (CD13 and CD14). All of these markers are used in consensus protocols and methods to immunoclassify hematopoietic malignancies (23,24) and their reasonable combination resulted in three working tubes: T1 (CD45-PECy5, CD34-APC, CD10-PE, CD19-FITC), T2 (CD7-APC, CD56-FITC, CD13-PE) and T3 (CD3-PE and CD14-FITC) (Supplemental Figure 1). Using this panel we were able to classify AL at least in the three groups according to the affected linage: B-ALL (77.88%), T-ALL (8.85%) and AML (13.27%) (Figures 1A-1B). Within the B-ALL group, three different clusters were identified depending on the differentiation stage of the leukemic blasts: ProB (CD34þCD10þCD19þ) (26.55%), PreB (CD34‒CD10þCD19þ) (19.47%) or the combination of both ProB and PreB precursors (31.86%) (Figure 1B). ProB-ALL Shows the Highest Content of Hematopoietic Stem and Progenitor Cells (HSPC) and the Best Production of NK Innate Cells A four-color panel was used to determine HSPC content in AL BM samples containing CD34-APC, Lin-PE, CD38FITC and CD45RA-PECy5. Hematopoietic stem cells (HSC) were defined as CD34þLin‒CD38‒CD45RA‒, whereas multi-lymphoid progenitors (MLP) as CD34þLin‒CD38‒CD45RAþ, multipotent progenitors (MPP) as CD34þLin‒CD38þCD45RA‒ and committed
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Figure 1. Classification and frequencies of childhood acute leukemias (AL). B-acute lymphoblastic leukemias (ALL) were appropriately subclassified in three groups according to the differentiation stages of the blasts within the bone marrow: ProB-ALL, PreB-ALL and ProB/PreB-ALL. T-ALL and AML were also distinguishable (A). Relative numbers of AL cases included in the study are shown (B). N 5 113. ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia. (A color figure can be found in the online version of this article.)
Normal Stem/Progenitor Cells in ALL
lymphoid progenitors (LP) as CD34þLin‒CD38þCD45RAþ (Figure 2A). We previously reported the reduced and dysfunctional content of HPSC (CD34þLin‒) in childhood B-ALL patients (15) and now notice that, from the stem cell pool to the earliest progenitors of the lymphoid program, a substantial reduction is apparent (Figure 2B). AL intergroup comparisons resulted in interesting differences in primitive cell contents. Whereas the lower content of HSPC was recorded in T-ALL BM (cell frequencies of 0.27%), ProB-ALL patients showed the highest relative numbers (3.51%) of all groups (Figure 3A), with no obvious differences in CD13þ aberrant-expressing ALL (Supplementary Figures 2Ae2E). Further subfractionation of the HSPC pool into MLP, MPP and LP confirmed that both HSC and MLP compartments are critically reduced in T-ALL and PreB patients (0.046 and 0.051%, respectively) and that the ProB entity has the highest amount of HSC, although never exceeding the normal values (2.5%) (Figure 3A). Data were not statistically different when comparing MPP and LP contents, but a slight tendency of T-ALL toward the lowest levels persists (Figure 3A). As expected and serving as group controls, ProB numbers were highest in ProB patients and the same was true for PreB cells in PreB patients, CD7þ cells in T-ALL patients and CD13 expression by AML patients. In all cases, almost 60% of BM cells were malignant blasts (Figure 3B). Neither CD3þ T lineage nor CD13 or CD14þ myeloid cells exhibited discrepancies among ALL subtypes (besides the CD3 expression by T-ALL leukemias and CD13 and CD14 by AML) (Figure 3C). Of high interest, frequency of CD56þ NK cells, especially the subset CD56þCD7þ in ProB patients, resulted remarkably distinct when compared with PreB subjects (Figure 3C). According to our previous CD13-associated pro-inflammatory microenvironment, a more detailed analysis indicated that ProB and ProB/PreB leukemias with aberrant expression of CD13 promote the production of more CD56þ NK cells (Supplementary Figure 2F). Normal HSPC Function Is Critically Damaged in T-ALL We previously reported a critical weakened lymphoid potential in the HSPC from global ALL patients and tested the lymphoid differentiation potential through fully controlled co-culture systems with MS-5 stromal feeders among the ALL subsets and upon cytometry purification of four primitive cell compartments. Despite the content of HSPC within ProB-ALL BM, they are not fully functional as total yield per input values did not show visible advantages with respect to other B-lineage ALL. In addition, lymphoid potential is apparently not affected in leukemic BM from individuals suffering CD13þ ProB-ALL (Figure 4A). In vitro analysis of T-ALL primitive cells confirmed an important functional damage analogous with
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their lack of HSPC. Furthermore, B-ALL BM are heterogeneous in cell cycle status and, after evaluation of the DNA content in the CD34þLin fraction, most ProB-ALL CD34þ cells are in G1/G0, whereas 40e50% of the counterparts in PreB-ALL are in synthesis phase (Figure 4B). CD45 has been considered as a powerful marker for abnormal hematopoietic cells in the BM. Within leukemic ProB-ALL BM, CD34þ compartment contained massive CD45dim cells and a scarce CD45hi population. After sorting and co-culturing on stromal monolayer feeders, total yield per input values revealed that the high normal lymphoid potential is kept in CD45hi cells (Figure 4C). Immunoscore Classified PreB- and T-ALL As Poor Prognosis Childhood Leukemias We performed Pearson’s correlation analysis among different subtypes of AL and contents of immune populations, confirming that T-ALL manifests strongly negative correlations in the immunological context (Figures 5Ae5C). As expected, a positive correlation tendency between frequency of mature immune cells and HPSC was displayed (Figures 5Fe5J). Thus, poor prognosis in T-ALL may relate to a debilitated hematopoiesis and ineffective immune surveillance. High Frequencies of HSPC Correlate with Standard-Risk Status in ProB-ALL Patients The sum of prognostic indicators allows stratification of patients into risk groups to decide the best way to conduct treatments. Variables driving risk status include age, white blood cell counts, BM blast infiltration, chromosomal abnormalities or treatment response (4). In this study, 84% of the patients were classified as high-risk, with no apparent correlation between B-precursor cell leukemia subtype and risk stratification (Figure 6A and Supplementary Figure 4). Standard-risk AL patients were shown to have significantly more HSPC compared with the high-risk group (Figure 6A), but no statistically significant differences were certain when analyzing fractionated HSC or the early progenitors MMP, MLP and LP (Supplemental Figure 3). Among B-ALL subtypes, high levels of HSPC are capable of distinguishing only ProB patients with standard-risk but the same was not true for the other leukemia subtypes (Figure 6A). No differences in immune content and risk stratification were found (data not shown). Together our findings suggest that subclassification of ALL based on differentiation stage of the abnormal pathway may be clinically relevant due to their distinct stem and progenitor cell content as well as the abundance of newly produced immune cells. Whereas size and function of primitive BM compartments are tightly regulated and are responsible for long-term production of hematopoietic cells and immune surveillance elements (Figure 6B), the counterparts in malignant settings are substantially reduced especially when patients debut with T-ALL or
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Figure 2. (A) Hematopoietic stem and the earliest lymphoid-progenitor cells (HSPC) in leukemic bone marrow are critically reduced. HSPC population was identified in the CD34þLin‒ compartment and subfractionated into HSC, MLP, MPP and LP to determine cell frequencies. (B) NBM 5 3, ALL 5 20. HSC, hematopoietic stem cell; MLP, multi-lymphoid progenitor; MPP, multipotent progenitor; LP, lymphoid progenitor; NBM, normal bone marrow; ALL, acute lymphoblastic leukemia.
PreB lineage leukemia (Figure 6B, lower panel). In contrast, ProB-ALL might be endowed with the highest relative contents of HSPC, a property that correlates to standard-risk prognosis at initial diagnosis (Figure 6B, middle panel).
Discussion AL represents a heterogeneous group of oligoclonal hematopoietic disorders and the most common childhood cancer worldwide (1e4). Most clinical manifestations are
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Figure 3. Abundance of hematopoietic stem and early progenitor cells (HSPC) and immune cell populations distinguish the ProB-ALL among leukemic subtypes. Frequencies of populations are expressed in percentage of bone marrow white blood cells (% of BM WBC): (A) HSPC; HSC; MLP; MPP; LP; (B) ProB cells; PreB cells; CD7þ cells; CD13þ cells; (C) CD3þ cells; CD14þ cells; CD56þ cells; CD56þCD7‒ cells and CD56þCD7þ cells. ProB 5 30, PreB 5 22, ProB/PreB 5 36, T-ALL 5 10, AML 5 15. HSC, hematopoietic stem cell; MLP, multi-lymphoid progenitor; MPP, multipotent progenitor; LP, lymphoid progenitor; NBM, normal bone marrow; ALL, acute lymphoblastic leukemia.
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Figure 4. Functional in vitro analysis of bone marrow progenitors reveals the altered hematopoietic compartment in T-ALL. Lymphoid differentiation potential was assessed through controlled co-culture systems on stromal cells where CD34þLin‒ population from patients was sorted and then co-cultured under lymphoid conditions. Newly produced cells were counted and yield per input was determined (A); DNA content gated on CD34þ cells was determined by flow cytometry and 7-AAD staining (B); CD45hi and CD45dim were sorted from CD34þLin population and then co-cultured with stromal feeders, after 4 weeks yield per input was determined (C). Each experiment was done in triplicate. (A color figure can be found in the online version of this article.)
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Figure 5. Correlation analysis of immune subsets and HSPC among acute leukemias. Per each subtype of leukemias, % of blast and % of CD56þ cells (A); % CD56þCD7þ cells (B); CD56þCD7‒ cells (C); CD3þ cells (D); CD14þ cells (E) and HSPC (FeJ) were determined. Y-axis was transformed through Ln function.
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Figure 6. High-risk acute leukemias have less hematopoietic stem and early progenitor cell (HSPC) contents. Risk stratification in the different B-acute leukemia subtypes (A). A proposal model indicating the utility of considering the pool size in BM of each primitive progenitor and their potential for cells of the immune surveillance (B). (A color figure can be found in the online version of this article.)
Normal Stem/Progenitor Cells in ALL
triggered by cytopenias (anemia, thrombocytopenia and neutropenia) (4) due to outcompetition for the same space in the BM (1,10). Unraveling the involved mechanisms is a topical issue (13,14,25,26). Delay in population dynamics knowledge is partially due to the complicated tracking of normal and malignant hematopoiesis in AL due to the associated intra- and inter-tumoral heterogeneity and the lack of surface or molecular markers to distinguish each entity (27). Following evolution of normal hematopoiesis within hematological malignancies should help to predict outcomes because leukemia is thought to start in BM in a very primitive population and hematopoietic progenitors are able to produce normal cells to preserve homeostasis. Favorable prognosis could then correlate with the healthy production of functional hematopoietic cells. As the immune system is also derived from hematopoietic stem cells (HSC), tumor immunosurveillance score may predict the capability of the immune system to eliminate or control malignant cells. Here we studied central control of hematopoiesis during AL, monitoring the content of very primitive populations and the rates of some immune cells in the BM at diagnosis. We designed a petite but very powerful antibody panel to distinguish and classify AL based on key surface markers for the lymphoid development and highly used for other groups (17,24). B-ALL is the most frequent childhood leukemia worldwide. Our study reflected its global epidemiology, followed by AML and T-ALL in frequency (23). Usually, blasts in B-cell leukemias are arrested in ProB and PreB stages (28) and we noted that one third of BALL patients have blasts in both populations (ProB/PreB) that may be further evidence of the oligoclonality of the disease (27). We determined the frequency of CD34þLin‒ in every BM sample; this fraction contains HSPC. Recently, we reported the critical reduction of normal HSPC in the leukemic BM and its impaired in vitro functionality (15). Surprisingly, subfractionation of early progenitors allowed us to see differences in the content of multi-lymphoid progenitors (MLP) and multi-potent progenitors (MPP) but not in HSC compartment or in the more committed lymphoid progenitors (LP) among leukemic patients. Currently, MLP is the earliest lymphoid progenitor (29). B-cells, Tcells, dendritic cells subsets, NK cells, lymphoid innate cells (LIC) and some marginal myeloid cells are derived from it. During murine T-ALL a reduction in the progenitor compartment has been reported, whereas HSC function was not remarkably compromised (16). Whether BM occupancy from leukemic or normal progenitors depends on the number of available leukemic/normal specialized niches in the BM is still a matter of debate (Balandran JC et al., work in progress). Subsequently, substantial differences in the HSPC, HSC and MLP contents among leukemic individuals were observed. Of note, T-ALL and PreB subtypes resulted to be less rich in this population at the central level, whereas ProB was distinguishably higher (15). CD13 expression
641
appeared to be very heterogeneous between individuals. Recently we reported that a subset of ALL patients creates a pro-inflammatory microenvironment driving the exhaustion of normal residual hematopoiesis in the BM of CD13þ expressing B-lineage leukemias (11). Interestingly, ProB-ALL seems to have significantly more T-cells in comparison to the ProB/PreB entity. Subfractioning of T-cell compartment should be imperative to explore Foxp3þ Tregs and other T-populations like CD8þ cells with important roles in cancer. It is known that stem cells and cancer cells can overexpress CD47 ‘‘don’t eat me’’ signal to evade phagocytosis (30), but CD14þ cells were not affected. NK cells are another cellular subset important to kill tumor cells (31). Data from several laboratories including ours have shown a decreased number of functional NK cells in the bloodstream as well as in the BM (32) (Vadillo E. et al., manuscript in preparation). Among leukemic subtypes, ProB has a special frequency of NK cells compared to PreB. Interestingly, more NK cell levels in ALL patients increase the success of the graft when transplanted. At the central level, multi-lymphoid progenitor (MLP) is the earliest NK progenitor and PreB individuals resulted to have the highest frequencies. Nevertheless, HSPC isolated from ProB patients cannot have high yields per input rates as we expected compared to Pre-B BM. Unpublished data from our laboratory confirmed that highly purified MLP from ALL patients can produce functional NK-like cells in vitro. Interestingly, ProB lymphoid potential resulted fully compromised in those patients with aberrant CD13 expression and also increased when CD34þ were sorted based on the CD45 expression. In contrast, B-ALL subtypes CD13þ have the highest frequencies of NK cells in the BM. Recent data from Vormoor’s laboratory indicate that clonally and functionality of primary ALL cells can be maintained in long-term co-cultures with Nestinþ mesenchymal stromal cells (MSC) (8) and leukemic support can be improved using MSC derived from the BM of ALL patients and 3D co-cultures (Balandran JC et al., 2016 submitted), highlighting the importance of leukemic-microenvironment intercommunication. In addition, LIC can co-exist in the HSPC compartment, disrupting the paradigmatic hematopoietic hierarchy (5,33). Leukemic and normal progenitors share the same spaces in the BM and normal niches should be occupied by HSPC followed by edition in favor of the tumor growth (13,25). Whether leukemic clones and normal HSPC have the same microenvironment requirements is still under debate; nevertheless, robust evidence suggests that leukemic cells are strongly dependent on their microenvironment. Novel study systems should be applied to board this question (1). Trying to unravel the exhaustion of HSPC pool in leukemic patients, our laboratory reported that pro-inflammatory cytokines produced by primary ALL cells lead proliferation in normal HSPC (11). Given a good hematopoietic and immunological scenario in the ProB group, higher levels of HSPC
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Balandran et al./ Archives of Medical Research 47 (2016) 629e643
can prevent leukemic progression as observed in CML and AML (18,19). Further experiments should be conducted under pre-clinical settings. In contrast, we confirmed that TALL is so aggressive due to lack of HSPC and immune cells to fight cancer. Further patient follow-up should be completed to predict clinical outcomes. Notably, immunoscore novel measurement incorporate the study of immune populations, particularly cytotoxic and memory T-cells in solid tumors. This criterion pointed to the notion to include immunological markers as a tool to predict and identify high-risk patients and clinical outcomes (20). BM-resident T cells include CD4þ and CD8þ populations in |1.5e2.5% of the total cells and antigen-mediated activation of T-cells has been vastly documented (34). In addition, LIC can hide in the immune system down-regulating many molecules (35). Importantly, the same immune mechanism involved to turn emergency hematopoiesis may play a role during leukemia progression because HPSC should remain protected to such immune activation (22). This work focused on the primitive hematopoietic content and immunological status in the BM of AL patients at diagnosis. The precise monitoring of patients throughout the disease and during remission can help to anticipate outcomes or to retrospectively predict the best candidates for BM transplant based on the immunosurveillance status. In conclusion, additional heterogeneity in AL results from the lineage differentiation stage at debut and contents of primitive hematopoietic stem and progenitor cells. HSPC abundance may aid to predict the clinical course of AL and to identify high-risk patients. A clearer understanding of their population dynamics and functional properties in the leukemia setting will potentially pave the way for targeted therapies.
Conflict of Interest The authors declare no conflict of interest.
Acknowledgments This work was supported by grants from the National Council of Science and Technology (CONACyT) (FOSISSS 2015-1-261848) and by the Mexican Institute for Social Security (FIS/IMSS/ PROT/G13/1229 and FIS/IMSS/PROT/G14/1289) to RP. EV and JCB acknowledge their scholarship from CONACyT and IMSS. Authors acknowledge the Flow Cytometry Core Facility from ‘‘Coordinacion de Investigacion en Salud’’ of IMSS.
Supplementary Data Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j. arcmed.2016.12.004.
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Normal Stem/Progenitor Cells in ALL 21. Fridman WH, Pages F, Sautes-Fridman C, Galon J. The immune contexture in human tumours: impact on clinical outcome. Nat Rev Cancer 2012;12:298e306. 22. Riether C, Sch€ urch CM, Ochsenbein AF. Regulation of hematopoietic and leukemic stem cells by the immune system. Cell Death Differ 2015;22:187e198. 23. Rothe G, Schmitz G. Consensus protocol for the flow cytometric immunophenotyping of hematopoietic malignancies. Working Group on Flow Cytometry and Image Analysis. Leukemia 1996;10:877e895. 24. Kalina T, Flores-Montero J, van der Velden VHJ, et al. EuroFlow standardization of flow cytometer instrument settings and immunophenotyping protocols. Leukemia 2012;26:1986e2010. 25. Enciso J, Mayani H, Mendoza L, Pelayo R. Modeling the pro-inflammatory tumor microenvironment in acute lymphoblastic leukemia predicts a breakdown of hematopoietic-mesenchymal communication networks. Front Physiol 2016;7:349. 26. van den Berk LCJ, van der Veer A, Willemse ME, et al. Disturbed CXCR4/CXCL12 axis in paediatric precursor B-cell acute lymphoblastic leukaemia. Br J Haematol 2014;166:240e249. 27. Vilchis-Ordo~ nez A, Dorantes-Acosta E, Vadillo E, Lopez-Martinez B, Pelayo R. Early hematopoietic differentiation in acute lymphoblastic leukemia: The interplay between leukemia-initiating cells and abnormal bone marrow microenvironment. In: Mejıa-Arangure JM, ed. Etiology of Acute Leukemias in Children. Springer International Publishing; 2016. pp. 291e320.
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28. Purizaca J, Meza I, Pelayo R. Early lymphoid development and microenvironmental cues in B-cell acute lymphoblastic leukemia. Arch Med Res 2012;43:89e101. 29. Doulatov S, Notta F, Eppert K, Nguyen LT, Ohashi PS, Dick JE. Revised map of the human progenitor hierarchy shows the origin of macrophages and dendritic cells in early lymphoid development. Nat Immunol 2010;11:585e593. 30. Chao MP, Alizadeh AA, Tang C, et al. Therapeutic antibody targeting of CD47 eliminates human acute lymphoblastic leukemia. Cancer Res 2011;71:1374e1384. 31. Cruz-Munoz M-E, Veillette A. Do NK cells always need a license to kill? Nat Immunol 2010;11:279e280. 32. Fei F, Lim M, George AA, et al. Cytotoxicity of CD56-positive lymphocytes against autologous B-cell precursor acute lymphoblastic leukemia cells. Leukemia 2015;29:788e797. 33. Rehe K, Wilson K, Bomken S, et al. Acute B lymphoblastic leukaemia-propagating cells are present at high frequency in diverse lymphoblast populations. EMBO Mol Med 2013;5: 38e51. 34. Monteiro JP, Benjamin A, Costa ES, Barcinski MA, Bonomo A. Normal hematopoiesis is maintained by activated bone marrow CD4þ T cells. Blood 2005;105:1484e1491. 35. Sch€urch CM, Riether C, Ochsenbein AF. Dendritic cell-based immunotherapy for myeloid leukemias. Front Immunol 2013;4: 496.
ORIGINAL ARTICLE
Analysis of Normal Hematopoietic Stem and Progenitor Cell Contents in Childhood Acute Leukemia Bone Marrow Juan Carlos Balandran,a,b,* Eduardo Vadillo,a,b,* David Dozal,c,* Alfonso Reyes-L opez,d c c Antonio Sandoval-Cabrera, Merle Denisse Laffont-Ortiz, Jessica L. Prieto-Chavez,e Armando Vilchis-Ordo~ nez,d Henry Quintela-Nu~ nez del Prado,f Hector Mayani,a g Juan Carlos N u~ nez-Enrıquez, Juan Manuel Mejıa-Arangure,g Briceida L opez-Martınez,d Elva Jimenez-Hernandez,h and Rosana Pelayoa a
Oncology Research Unit, Oncology Hospital, Instituto Mexicano del Seguro Social, Mexico City, Mexico b Departamento de Biomedicina Molecular, CINVESTAV, Mexico City, Mexico c Hospital para el Ni~no, Instituto Materno Infantil del Estado de Mexico, Mexico d Hospital Infantil de Mexico ‘‘Federico Gomez’’, Mexico City, Mexico e Unidad de Investigacion Medica en, Hospital de Especialidades, Instituto Mexicano del Seguro Social, Mexico City, Mexico f Unidad Medica de Alta Especialidad, ‘‘Dr. Victorio de la Fuente Narvaez’’, Instituto Mexicano del Seguro Social, Mexico City, Mexico g Unidad de Epidemiologıa de Investigacion Clınica Hospital de Pediatrıa, Instituto Mexicano del Seguro Social, Mexico City, Mexico h Hospital Pediatrico Moctezuma, Mexico City, Mexico Received for publication October 24, 2016; accepted November 23, 2016 (ARCMED-D-16-00651).
Background and Aims. Childhood acute leukemias (AL) are characterized by the excessive production of malignant precursor cells at the expense of effective blood cell development. The dominance of leukemic cells over normal progenitors may result in either direct suppression of functional hematopoiesis or remodeling of microenvironmental niches, contributing to BM failure and AL-associated mortality. We undertook this study to investigate the contents and functional activity of hematopoietic stem/progenitor cells (HSPC) and their relationship to immune cell production and risk status in AL pediatric patients. Methods. Multiparametric flow cytometry of BM aspirates was performed to classify AL on the basis of lineage and differentiation stages and to analyze HSPC and immune cell frequencies. Controlled co-culture systems were conducted to evaluate functional lineage potentials of primitive cells. Statistical correlations and inter-group significant differences were established. Results. Among 113 AL BM aspirates, 26.5% corresponded to ProB, 19.5% to PreB and 32% contain ProB and PreB differentiation stages, whereas nearly 9% of the cases were T- and 13% myeloid-lineage leukemias. We identified ProB-ALL as the subtype endowed with the highest relative contents of HSPC, whereas T-ALL and PreB-ALL showed a critically reduced size of both HSC and MLP compartments. Notably, lower cell frequencies of HSPC in ProB-ALL correlated to high-risk prognosis at disease debut. Conclusions. HSPC abundance at initial diagnosis may aid to predict the clinical course of ALL and to identify high-risk patients. A clearer understanding of their population dynamics and functional properties in the leukemia setting will potentially pave the way for targeted therapies. Ó 2016 IMSS. Published by Elsevier Inc. Key Words: Acute lymphoblastic leukemia, Stem/progenitor cells, Lymphoid immune cells, Bone marrow, ProB-ALL high risk.
*
These authors contributed equally to this work. Address reprint requests to: Dr. Rosana Pelayo, Oncology Research Unit, Oncology Hospital, IMSS, Av. Cuauhtemoc 330, Colonia Doctores,
06720 Mexico City, Mexico; Phone: (þ52) (55) 5627-6915 ext. 22705 or 22710; FAX: NEEDED; E-mail: [email protected].
0188-4409/$ - see front matter. Copyright Ó 2016 IMSS. Published by Elsevier Inc. http://dx.doi.org/10.1016/j.arcmed.2016.12.004
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Introduction The most important disease endangering the hematopoietic system in pediatric patients is acute leukemia (AL), which constitutes the major cause of death worldwide (1,2). Particularly, childhood AL in Mexico shows among the highest incidence rates with extraordinary heterogeneity and inferior outcomes (3). Almost 85% of the cases compromise the lymphoid linage, whereas 15e20% are from myeloid origins (2). Historically, T-lineage leukemias have been considered the subtype endowed with the most adverse scores (4). However, accumulating evidence of cytopenias and impaired hematopoiesis in some B- and myeloid-AL, often resulting in bone marrow (BM) failure and fatal outcomes has suggested that ineffective hematopoiesis is the clinical hallmark of all AL (4). The high proliferative activity and oligoclonal dominance of leukemic cells over the normal hematopoietic stem and progenitor cells (HSPC) displace them in a dynamic competition for the BM niches (1). As leukemic-initiating cells (LICs) coexist with normal primitive cells within the CD34þ cell compartment, no surface markers can be used so far to distinguish normal HSPC from LICs, making the normal vs. malignant population dynamics still uncertain (5). Hematopoietic primitive cells fulfill their functional production of blood cells in the context of microenvironmental regulation. Emerging research sustains the notion that neoplastic cells are also dependent on the surrounding microenvironment and point to the influence of specialized niches for leukemia progression (6e9). Hematopoietic suppression as a result of excessive leukemic growth may be contributed by direct inhibition of developing cells by soluble factors, by the outcompetition for stem cell niches and by leukemic cell-induced microenvironment remodeling, creating a leukemic sanctuary unsuitable for normal hematopoiesis (10). The biological and molecular mechanisms involved in the displacement of normal hematopoiesis are currently under investigation by a number of groups. Our recent findings suggest that primary B-lineage ALL cells are capable of producing pro-inflammatory cytokines and abnormal amounts of growth factors that interfere with regular hematopoietic proliferation and differentiation processes (11). Moreover, exhaustion of the HSPC compartment has been demonstrated in chronic myeloid leukemia (CML) in a leukemic cell-derived IL-6 dependent manner (12). Elegant animal models have been useful to confirm that leukemic cells hijack and remodel normal mouse niches displacing normal hematopoiesis to favor leukemic progression concomitant to down-regulation of the primary chemokine CXCL12 in BM (13,14). Accordingly, we have reported that cell frequencies of functional HSPC are critically reduced in B-ALL patients (15), with our recent data suggesting that mesenchymal reticular niche within the leukemic BM is not able to sustain ex vivo
normal lymphopoiesis due to a diminished expression of CXCL12, but facilitating instead the maintenance and progression of primary ALL cells (Balandran JC et al., submitted). Of note, abnormal BM mesenchymal stromal cell (MSC) niches in a T-ALL mice model were shown to be unable to support HSPC due to their accelerated senescence (16). Strikingly, minimal residual disease (MRD) detection has been the most important prognostic indicator of treatment success or failure, with the supposed concomitant emergence of normal hematopoiesis responsible for repopulating the BM following remission (17). To date, no reports monitoring HSPC numbers and function in childhood AL or correlation studies with clinical outcomes are recorded. Rebounding of normal hematopoiesis should be reflected in blood and BM cell frequencies close to homeostatic values. Interestingly, experimental data from a competitive repopulation in a pre-clinical model showed a good correlation of HSPC with good prognosis, with increasing doses of normal HSPC delaying the LIC establishment (18). Similar observations have resulted from experimental models of AML (19). Mature B, T, NK and dendritic cells as well as macrophages are a substantial part of the BM microenvironment. As all immune cells derive from HSPC, the evaluation of alterations in such a primitive compartment is of high relevance. In solid tumor settings, immune cell contents constitute a prognosis factor where global outcomes depend on immunoregulatory cell subsets abundance. Further immunity evaluation in cancer will provide better information with prognostic value (20). A novel immunoscore allows classification of malignancies within an immune context where, remarkably, infiltration of subsets of T cells has been associated with good prognosis in several solid tumors (21), whereas activation of T cells in leukemias contribute to tumor surveillance and control (22). In this work the contents and functional activity of HSPC and their relationship to immune cell production and risk status in acute leukemia have been investigated. Among five subtypes of AL (ProB-ALL, PreB-ALL, ProB/PreB-ALL, T-ALL and AML), we found that ProBALL has the highest contents of HSPC, whereas T-ALL and PreB-ALL showed scarce HSC and MLP cells. Notably, lower cell frequencies of HSPC in ProB-ALL correlated to high-risk prognosis. To the best of our knowledge, this is the first report describing BM normal hematopoietic content in the immune context in childhood AL.
Materials and Methods Patient Characteristics and Sample Collection One hundred thirteen children diagnosed with ALL were included in this study: 63 were referred to the ‘‘Hospital para
Normal Stem/Progenitor Cells in ALL
631
Table 1. Patient characteristics Patient 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Age (years) 9 11 9 10 9 8 10 3 14 16 11 15 8 9 14 14 12 8 3 2 9 4 9 9 8 11 10 9 10 15 13 7 8 7 14 7 8 10 3 15 14 5 10 7 12 8 3 4 4 7 11 6 14 14 9 9 5 9 8 9
Sex
Phenotype
M F M M F M M F M M M M M M M M M M F F M M M F F M M F M M F F M M M F F M F F F F F M F F F F F M F F F M F F F F F F
AML AML AML AML AML AML AML AML AML AML AML AML AML AML AML PreB PreB PreB PreB PreB PreB PreB PreB PreB PreB PreB PreB PreB PreB PreB PreB PreB PreB PreB PreB PreB PreB ProB ProB ProB ProB ProB ProB ProB ProB ProB ProB ProB ProB ProB ProB ProB ProB ProB ProB ProB ProB ProB ProB ProB
WBC 3103/mm3
BM Blast (%)
Chromosomal aberrations
Risk
10.5 32 52 45.6 134 129.1 62.8 39.6 59.3 18.5 52.5 45 2.2 187 5.1 293.4 7.1 281 123 188 23 143 121 109 71.7 45.6 112 108.6 110.6 110.8 12.5 104.9 58.3 108.3 53.5 164.5 107.9 4.9 5.1 118.9 58.6 47.9 285.5 34 45.6 78.5 121.4 112 60.4 83.51 -
5 74.9 75 23.6 87 -
Neg t (15,17)(q24;q21) Neg t (15,17)(q24;q21) PML ex3-RARA ex5 t (15,17)(q22;q21) PML ex3-RARA ex5 Neg Neg t (9,22)(q34;q11) BCR ex13 - ABL1 ex2 (b2a2) € ı PBX ex3 t (1,19)(q23;p13) TCF3 ex16, A Neg Neg t (15,17)(q22;q21) PML ex3-RARA ex5 t (12,21)(p13;q22) ETV6 ex5-RUNX1 ex4: t (9,22)(q34;q11) Neg t (1,19)(q23;p13)TCF3-PBX1(PRL) Neg Neg t (12,21)(p13;q22) ETV6 ex5-RUNX1 ex3 Neg Neg Neg Neg -
High High High High High High High High High High High High High High High High High High High High High High High High High High High High High High Standard High Standard Standard High High High High High Standard High High High Standard High High High High Standard High High Standard High High High High Standard High High High
45 62 70.3 65.6 97 0.07 0.07 92.33 70 94.6 67 89 73.5 48.3 67 56 78 67 90.4 98.6 78.6 94.6 56.4 84 0.03 82.53 74 67 87.8 85 80.6 72 70 92.3 83.1 90.8 90 12 65 80.02 78 56 49.5 83.51 -
(continued on next page)
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Table 1 (continued ) Patient 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113
Age (years) 16 5 15 7 8 7 13 8 7 14 9 11 3 7 2 4 3 2 8m 12 3 9 16 4 3 1 3 7 5 6 6 15 5 4 6 2 5 6 14 7 2 2 1 3 13 12 5 15 14 15 3 2 5
Sex
Phenotype
WBC 3103/mm3
BM Blast (%)
Chromosomal aberrations
Risk
M F F M M M F F F M M M M F M F F F M F F M M M F F M M M F M F M M F F F F M M F F M M F M F F F F M M F
ProB ProB ProB ProB ProB ProB ProB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB ProB/PreB T-ALL T-ALL T-ALL T-ALL T-ALL T-ALL T-ALL T-ALL T-ALL T-ALL
45.7 34.5 29 89.4 60.3 48.6 117 62.3 100.7 270 119.2 33.9 160.5 37.5 176.3 45.6 125.3 25.6 150 55 9.5 64.6 123 16.5 162.5 45.3 12.4 27.6 26.9 29.4 145.2 43.2 134.5 40.6 15.6 23.5 45.2 59.9 42.1 135.6 791 116 316.2 6.4 79.4 107.3 112.1 231.5
28.5 86.4 83.74 70 81.01 80 71.23 88.5 81 85.9 83 92.2 86.35 87 76.89 90.6 93.88 62.4 93.9 86.6 74.9 89.7 66 70 95 65.4 37.8 68.4 78.6 76.1 67.8 25 89.4 71.37 88.05 76.4 22.8 91.47 82.24 67.3 96.06 86 88 30 87 94.1 89.5 76.4
Neg Neg t (15,17)(q24;q21) Neg t (1,19)(q23;p13)TCF3-PBX1(PRL) Neg Neg t (1,19)(q23;p13) t (12,21)(p13;q22) ETV6 ex5-RUNX1 ex4: Neg Neg Neg t (1,19)(q23;p13)TCF3-PBX1(PRL) Neg t (12,21)(p13;q22) ETV6 ex5-RUNX1 ex4: € ı ERG ex13 t (16,21)(p11;q22) FUS ex7, A t (12,21)(p13;q22) ETV6 ex5-RUNX1 ex4: Neg Neg Neg Neg t (12,21)(p13;q22) Neg Del TAL1 Neg Neg -
High Standard High High High High High High High High High High High High High High High High High High High High High High High High High Standard Standard Standard Standard High Standard High Standard High Standard Standard High High High High High High High High High High High High High High High
el Ni~ no’’, 43 to the ‘‘Hospital Pediatrico Moctezuma’’ and seven to the ‘‘Hospital Infantil de Mexico Federico Gomez’’. Among them, 84.0% were stratified for high-risk disease, whereas 15.9% were stratified for standard-risk according to the blast cell count, age, T-linage and/or genetics criteria; 49.5% of the patients were male and 50.4% were female.
Median age values were 8 years (8 monthse16 years) and 3 years (1e7 years) for the high-risk and standard-risk group, respectively (Table 1). Control samples were obtained from healthy children undergoing minor orthopedic surgery BM specimens were collected by aspiration before any treatment, respecting international and institutional guidelines.
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633
All procedures were approved by the Ethics, Research and Biosafety Committee of the Hospitals and by the National Committee of Scientific Research at the Mexican Institute for Social Security (Registries CIEICE-007-01-13, R-20123602-29 and R-2015-785-120). All samples were collected after written informed consent from parents.
Cells were classified into phases of cell cycle by using the Cell Cycle Flow Cytometry Analysis Software (FlowJo, Tree Star, Inc.), which calculates the cell frequencies in G1/G0, S and G2/M.
Immunophenotyping by Flow Cytometry
Data were analyzed using STATA v.10.0 (Stata Corporation) and Prism V3.02 (GraphPad) softwares. Differences within groups were established by non-parametric test, considering significant probability values ! 0.05.
White blood cell numbers in BM samples were determined using Turk’s solution and 5 105 leukocytes were added per tube. Cells were stained with hCD45-PE-Cy5, hCD34-APC, hCD19FITC and hCD10-PE (Tube 1). The same cell number was stained with hCD7-APC, hCD56-FITC and hCD13-PE (Tube 2) or with hCD3-PE and hCD14-FITC (Tube 3) (Supplementary Figure 1). In addition, 1 106 leukocytes were stained with a lineage antibody cocktail (Lin: hCD3-PE, hCD8PE, hCD11c-PE, hCD14-PE, hCD19-PE, hCD56-PE and hCD235a-PE), hCD34-APC, hCD38-FITC and hCD45RAPE-Cy5. All antibodies were from BioLegend, San Diego, CA. Red blood cells were lysed with BD pharm lyse (BD Biosciences, San Jose, CA) following antibody staining. Samples were acquired on a CyAn (Beckman Coulter, Pasadena, CA) or a BD FACSCanto II cytometers. Analysis of flow cytometry data was performed using the FlowJo 10.0.8 software. Isolation of HSPC Mononuclear cells (MNC) from BM samples were prepared by using Ficoll-Paque Plus (GE Healthcare Bioscience, Uppsala, Sweden) and CD34þ pool was enriched from MNC with the human CD34 progenitor cell isolation kit (Miltenyi Biotec, San Diego, CA) according to the manufacturer’s instructions. CD34þ fraction was stained with anti-CD34 and anti-lineage antibodies followed by HSPC sorting on the basis of CD34 expression and lacking of linage markers (CD34þLin‒), using a BD FACSAria sorter. Samples were individually manipulated and no sample pooling was performed for any of the conducted experiments. Co-cultures in Lymphoid Conditions HSPC were co-cultured with MS-5 stromal cells for 30 day in triplicate in a-MEM (Gibco, Anaheim, CA) culture medium supplemented with 10% fetal bovine serum (Gibco), 1 ng/mL Flt3-L, 2 ng/mL SCF, 5 ng/mL IL-7, and 10 ng/ mL IL-15 (PeproTech, Rocky Hill, NJ) and contained 100 U/mL penicillin and 100 U/mL streptomycin (Gibco). Cocultures were incubated at 37 C in a humidified atmosphere of 5% of CO2. At the end of the cultures, cells were harvested and viability was also determined. Cell Cycle Analysis After membrane staining with anti-hCD34 and a lineage antibody cocktail, cells were fixed and permeabilized prior to 7-aminoactinomycin (7-ADD) staining (BD Biosciences).
Statistics
Research on Human Subjects As stated, all procedures were approved by the Ethics, Research and Biosafety Committee of the Hospitals and by the National Committee of Scientific Research at the Mexican Institute for Social Security (Registries CIEICE-007-01-13, R-2012-3602-29 and R-2015-785-120). All samples were collected after written informed consent from the parents.
Results Multiparametric Flow Cytometry Immunophenotyping and Classification of Childhood Acute Leukemias We designed a panel based on the lymphoid differentiation surface markers (CD45, CD34, CD10, CD19 and CD7), additional immune lymphoid cells (CD56 and CD3) and myeloid markers (CD13 and CD14). All of these markers are used in consensus protocols and methods to immunoclassify hematopoietic malignancies (23,24) and their reasonable combination resulted in three working tubes: T1 (CD45-PECy5, CD34-APC, CD10-PE, CD19-FITC), T2 (CD7-APC, CD56-FITC, CD13-PE) and T3 (CD3-PE and CD14-FITC) (Supplemental Figure 1). Using this panel we were able to classify AL at least in the three groups according to the affected linage: B-ALL (77.88%), T-ALL (8.85%) and AML (13.27%) (Figures 1A-1B). Within the B-ALL group, three different clusters were identified depending on the differentiation stage of the leukemic blasts: ProB (CD34þCD10þCD19þ) (26.55%), PreB (CD34‒CD10þCD19þ) (19.47%) or the combination of both ProB and PreB precursors (31.86%) (Figure 1B). ProB-ALL Shows the Highest Content of Hematopoietic Stem and Progenitor Cells (HSPC) and the Best Production of NK Innate Cells A four-color panel was used to determine HSPC content in AL BM samples containing CD34-APC, Lin-PE, CD38FITC and CD45RA-PECy5. Hematopoietic stem cells (HSC) were defined as CD34þLin‒CD38‒CD45RA‒, whereas multi-lymphoid progenitors (MLP) as CD34þLin‒CD38‒CD45RAþ, multipotent progenitors (MPP) as CD34þLin‒CD38þCD45RA‒ and committed
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Figure 1. Classification and frequencies of childhood acute leukemias (AL). B-acute lymphoblastic leukemias (ALL) were appropriately subclassified in three groups according to the differentiation stages of the blasts within the bone marrow: ProB-ALL, PreB-ALL and ProB/PreB-ALL. T-ALL and AML were also distinguishable (A). Relative numbers of AL cases included in the study are shown (B). N 5 113. ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia. (A color figure can be found in the online version of this article.)
Normal Stem/Progenitor Cells in ALL
lymphoid progenitors (LP) as CD34þLin‒CD38þCD45RAþ (Figure 2A). We previously reported the reduced and dysfunctional content of HPSC (CD34þLin‒) in childhood B-ALL patients (15) and now notice that, from the stem cell pool to the earliest progenitors of the lymphoid program, a substantial reduction is apparent (Figure 2B). AL intergroup comparisons resulted in interesting differences in primitive cell contents. Whereas the lower content of HSPC was recorded in T-ALL BM (cell frequencies of 0.27%), ProB-ALL patients showed the highest relative numbers (3.51%) of all groups (Figure 3A), with no obvious differences in CD13þ aberrant-expressing ALL (Supplementary Figures 2Ae2E). Further subfractionation of the HSPC pool into MLP, MPP and LP confirmed that both HSC and MLP compartments are critically reduced in T-ALL and PreB patients (0.046 and 0.051%, respectively) and that the ProB entity has the highest amount of HSC, although never exceeding the normal values (2.5%) (Figure 3A). Data were not statistically different when comparing MPP and LP contents, but a slight tendency of T-ALL toward the lowest levels persists (Figure 3A). As expected and serving as group controls, ProB numbers were highest in ProB patients and the same was true for PreB cells in PreB patients, CD7þ cells in T-ALL patients and CD13 expression by AML patients. In all cases, almost 60% of BM cells were malignant blasts (Figure 3B). Neither CD3þ T lineage nor CD13 or CD14þ myeloid cells exhibited discrepancies among ALL subtypes (besides the CD3 expression by T-ALL leukemias and CD13 and CD14 by AML) (Figure 3C). Of high interest, frequency of CD56þ NK cells, especially the subset CD56þCD7þ in ProB patients, resulted remarkably distinct when compared with PreB subjects (Figure 3C). According to our previous CD13-associated pro-inflammatory microenvironment, a more detailed analysis indicated that ProB and ProB/PreB leukemias with aberrant expression of CD13 promote the production of more CD56þ NK cells (Supplementary Figure 2F). Normal HSPC Function Is Critically Damaged in T-ALL We previously reported a critical weakened lymphoid potential in the HSPC from global ALL patients and tested the lymphoid differentiation potential through fully controlled co-culture systems with MS-5 stromal feeders among the ALL subsets and upon cytometry purification of four primitive cell compartments. Despite the content of HSPC within ProB-ALL BM, they are not fully functional as total yield per input values did not show visible advantages with respect to other B-lineage ALL. In addition, lymphoid potential is apparently not affected in leukemic BM from individuals suffering CD13þ ProB-ALL (Figure 4A). In vitro analysis of T-ALL primitive cells confirmed an important functional damage analogous with
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their lack of HSPC. Furthermore, B-ALL BM are heterogeneous in cell cycle status and, after evaluation of the DNA content in the CD34þLin fraction, most ProB-ALL CD34þ cells are in G1/G0, whereas 40e50% of the counterparts in PreB-ALL are in synthesis phase (Figure 4B). CD45 has been considered as a powerful marker for abnormal hematopoietic cells in the BM. Within leukemic ProB-ALL BM, CD34þ compartment contained massive CD45dim cells and a scarce CD45hi population. After sorting and co-culturing on stromal monolayer feeders, total yield per input values revealed that the high normal lymphoid potential is kept in CD45hi cells (Figure 4C). Immunoscore Classified PreB- and T-ALL As Poor Prognosis Childhood Leukemias We performed Pearson’s correlation analysis among different subtypes of AL and contents of immune populations, confirming that T-ALL manifests strongly negative correlations in the immunological context (Figures 5Ae5C). As expected, a positive correlation tendency between frequency of mature immune cells and HPSC was displayed (Figures 5Fe5J). Thus, poor prognosis in T-ALL may relate to a debilitated hematopoiesis and ineffective immune surveillance. High Frequencies of HSPC Correlate with Standard-Risk Status in ProB-ALL Patients The sum of prognostic indicators allows stratification of patients into risk groups to decide the best way to conduct treatments. Variables driving risk status include age, white blood cell counts, BM blast infiltration, chromosomal abnormalities or treatment response (4). In this study, 84% of the patients were classified as high-risk, with no apparent correlation between B-precursor cell leukemia subtype and risk stratification (Figure 6A and Supplementary Figure 4). Standard-risk AL patients were shown to have significantly more HSPC compared with the high-risk group (Figure 6A), but no statistically significant differences were certain when analyzing fractionated HSC or the early progenitors MMP, MLP and LP (Supplemental Figure 3). Among B-ALL subtypes, high levels of HSPC are capable of distinguishing only ProB patients with standard-risk but the same was not true for the other leukemia subtypes (Figure 6A). No differences in immune content and risk stratification were found (data not shown). Together our findings suggest that subclassification of ALL based on differentiation stage of the abnormal pathway may be clinically relevant due to their distinct stem and progenitor cell content as well as the abundance of newly produced immune cells. Whereas size and function of primitive BM compartments are tightly regulated and are responsible for long-term production of hematopoietic cells and immune surveillance elements (Figure 6B), the counterparts in malignant settings are substantially reduced especially when patients debut with T-ALL or
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Figure 2. (A) Hematopoietic stem and the earliest lymphoid-progenitor cells (HSPC) in leukemic bone marrow are critically reduced. HSPC population was identified in the CD34þLin‒ compartment and subfractionated into HSC, MLP, MPP and LP to determine cell frequencies. (B) NBM 5 3, ALL 5 20. HSC, hematopoietic stem cell; MLP, multi-lymphoid progenitor; MPP, multipotent progenitor; LP, lymphoid progenitor; NBM, normal bone marrow; ALL, acute lymphoblastic leukemia.
PreB lineage leukemia (Figure 6B, lower panel). In contrast, ProB-ALL might be endowed with the highest relative contents of HSPC, a property that correlates to standard-risk prognosis at initial diagnosis (Figure 6B, middle panel).
Discussion AL represents a heterogeneous group of oligoclonal hematopoietic disorders and the most common childhood cancer worldwide (1e4). Most clinical manifestations are
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Figure 3. Abundance of hematopoietic stem and early progenitor cells (HSPC) and immune cell populations distinguish the ProB-ALL among leukemic subtypes. Frequencies of populations are expressed in percentage of bone marrow white blood cells (% of BM WBC): (A) HSPC; HSC; MLP; MPP; LP; (B) ProB cells; PreB cells; CD7þ cells; CD13þ cells; (C) CD3þ cells; CD14þ cells; CD56þ cells; CD56þCD7‒ cells and CD56þCD7þ cells. ProB 5 30, PreB 5 22, ProB/PreB 5 36, T-ALL 5 10, AML 5 15. HSC, hematopoietic stem cell; MLP, multi-lymphoid progenitor; MPP, multipotent progenitor; LP, lymphoid progenitor; NBM, normal bone marrow; ALL, acute lymphoblastic leukemia.
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Figure 4. Functional in vitro analysis of bone marrow progenitors reveals the altered hematopoietic compartment in T-ALL. Lymphoid differentiation potential was assessed through controlled co-culture systems on stromal cells where CD34þLin‒ population from patients was sorted and then co-cultured under lymphoid conditions. Newly produced cells were counted and yield per input was determined (A); DNA content gated on CD34þ cells was determined by flow cytometry and 7-AAD staining (B); CD45hi and CD45dim were sorted from CD34þLin population and then co-cultured with stromal feeders, after 4 weeks yield per input was determined (C). Each experiment was done in triplicate. (A color figure can be found in the online version of this article.)
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Figure 5. Correlation analysis of immune subsets and HSPC among acute leukemias. Per each subtype of leukemias, % of blast and % of CD56þ cells (A); % CD56þCD7þ cells (B); CD56þCD7‒ cells (C); CD3þ cells (D); CD14þ cells (E) and HSPC (FeJ) were determined. Y-axis was transformed through Ln function.
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Figure 6. High-risk acute leukemias have less hematopoietic stem and early progenitor cell (HSPC) contents. Risk stratification in the different B-acute leukemia subtypes (A). A proposal model indicating the utility of considering the pool size in BM of each primitive progenitor and their potential for cells of the immune surveillance (B). (A color figure can be found in the online version of this article.)
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triggered by cytopenias (anemia, thrombocytopenia and neutropenia) (4) due to outcompetition for the same space in the BM (1,10). Unraveling the involved mechanisms is a topical issue (13,14,25,26). Delay in population dynamics knowledge is partially due to the complicated tracking of normal and malignant hematopoiesis in AL due to the associated intra- and inter-tumoral heterogeneity and the lack of surface or molecular markers to distinguish each entity (27). Following evolution of normal hematopoiesis within hematological malignancies should help to predict outcomes because leukemia is thought to start in BM in a very primitive population and hematopoietic progenitors are able to produce normal cells to preserve homeostasis. Favorable prognosis could then correlate with the healthy production of functional hematopoietic cells. As the immune system is also derived from hematopoietic stem cells (HSC), tumor immunosurveillance score may predict the capability of the immune system to eliminate or control malignant cells. Here we studied central control of hematopoiesis during AL, monitoring the content of very primitive populations and the rates of some immune cells in the BM at diagnosis. We designed a petite but very powerful antibody panel to distinguish and classify AL based on key surface markers for the lymphoid development and highly used for other groups (17,24). B-ALL is the most frequent childhood leukemia worldwide. Our study reflected its global epidemiology, followed by AML and T-ALL in frequency (23). Usually, blasts in B-cell leukemias are arrested in ProB and PreB stages (28) and we noted that one third of BALL patients have blasts in both populations (ProB/PreB) that may be further evidence of the oligoclonality of the disease (27). We determined the frequency of CD34þLin‒ in every BM sample; this fraction contains HSPC. Recently, we reported the critical reduction of normal HSPC in the leukemic BM and its impaired in vitro functionality (15). Surprisingly, subfractionation of early progenitors allowed us to see differences in the content of multi-lymphoid progenitors (MLP) and multi-potent progenitors (MPP) but not in HSC compartment or in the more committed lymphoid progenitors (LP) among leukemic patients. Currently, MLP is the earliest lymphoid progenitor (29). B-cells, Tcells, dendritic cells subsets, NK cells, lymphoid innate cells (LIC) and some marginal myeloid cells are derived from it. During murine T-ALL a reduction in the progenitor compartment has been reported, whereas HSC function was not remarkably compromised (16). Whether BM occupancy from leukemic or normal progenitors depends on the number of available leukemic/normal specialized niches in the BM is still a matter of debate (Balandran JC et al., work in progress). Subsequently, substantial differences in the HSPC, HSC and MLP contents among leukemic individuals were observed. Of note, T-ALL and PreB subtypes resulted to be less rich in this population at the central level, whereas ProB was distinguishably higher (15). CD13 expression
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appeared to be very heterogeneous between individuals. Recently we reported that a subset of ALL patients creates a pro-inflammatory microenvironment driving the exhaustion of normal residual hematopoiesis in the BM of CD13þ expressing B-lineage leukemias (11). Interestingly, ProB-ALL seems to have significantly more T-cells in comparison to the ProB/PreB entity. Subfractioning of T-cell compartment should be imperative to explore Foxp3þ Tregs and other T-populations like CD8þ cells with important roles in cancer. It is known that stem cells and cancer cells can overexpress CD47 ‘‘don’t eat me’’ signal to evade phagocytosis (30), but CD14þ cells were not affected. NK cells are another cellular subset important to kill tumor cells (31). Data from several laboratories including ours have shown a decreased number of functional NK cells in the bloodstream as well as in the BM (32) (Vadillo E. et al., manuscript in preparation). Among leukemic subtypes, ProB has a special frequency of NK cells compared to PreB. Interestingly, more NK cell levels in ALL patients increase the success of the graft when transplanted. At the central level, multi-lymphoid progenitor (MLP) is the earliest NK progenitor and PreB individuals resulted to have the highest frequencies. Nevertheless, HSPC isolated from ProB patients cannot have high yields per input rates as we expected compared to Pre-B BM. Unpublished data from our laboratory confirmed that highly purified MLP from ALL patients can produce functional NK-like cells in vitro. Interestingly, ProB lymphoid potential resulted fully compromised in those patients with aberrant CD13 expression and also increased when CD34þ were sorted based on the CD45 expression. In contrast, B-ALL subtypes CD13þ have the highest frequencies of NK cells in the BM. Recent data from Vormoor’s laboratory indicate that clonally and functionality of primary ALL cells can be maintained in long-term co-cultures with Nestinþ mesenchymal stromal cells (MSC) (8) and leukemic support can be improved using MSC derived from the BM of ALL patients and 3D co-cultures (Balandran JC et al., 2016 submitted), highlighting the importance of leukemic-microenvironment intercommunication. In addition, LIC can co-exist in the HSPC compartment, disrupting the paradigmatic hematopoietic hierarchy (5,33). Leukemic and normal progenitors share the same spaces in the BM and normal niches should be occupied by HSPC followed by edition in favor of the tumor growth (13,25). Whether leukemic clones and normal HSPC have the same microenvironment requirements is still under debate; nevertheless, robust evidence suggests that leukemic cells are strongly dependent on their microenvironment. Novel study systems should be applied to board this question (1). Trying to unravel the exhaustion of HSPC pool in leukemic patients, our laboratory reported that pro-inflammatory cytokines produced by primary ALL cells lead proliferation in normal HSPC (11). Given a good hematopoietic and immunological scenario in the ProB group, higher levels of HSPC
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can prevent leukemic progression as observed in CML and AML (18,19). Further experiments should be conducted under pre-clinical settings. In contrast, we confirmed that TALL is so aggressive due to lack of HSPC and immune cells to fight cancer. Further patient follow-up should be completed to predict clinical outcomes. Notably, immunoscore novel measurement incorporate the study of immune populations, particularly cytotoxic and memory T-cells in solid tumors. This criterion pointed to the notion to include immunological markers as a tool to predict and identify high-risk patients and clinical outcomes (20). BM-resident T cells include CD4þ and CD8þ populations in |1.5e2.5% of the total cells and antigen-mediated activation of T-cells has been vastly documented (34). In addition, LIC can hide in the immune system down-regulating many molecules (35). Importantly, the same immune mechanism involved to turn emergency hematopoiesis may play a role during leukemia progression because HPSC should remain protected to such immune activation (22). This work focused on the primitive hematopoietic content and immunological status in the BM of AL patients at diagnosis. The precise monitoring of patients throughout the disease and during remission can help to anticipate outcomes or to retrospectively predict the best candidates for BM transplant based on the immunosurveillance status. In conclusion, additional heterogeneity in AL results from the lineage differentiation stage at debut and contents of primitive hematopoietic stem and progenitor cells. HSPC abundance may aid to predict the clinical course of AL and to identify high-risk patients. A clearer understanding of their population dynamics and functional properties in the leukemia setting will potentially pave the way for targeted therapies.
Conflict of Interest The authors declare no conflict of interest.
Acknowledgments This work was supported by grants from the National Council of Science and Technology (CONACyT) (FOSISSS 2015-1-261848) and by the Mexican Institute for Social Security (FIS/IMSS/ PROT/G13/1229 and FIS/IMSS/PROT/G14/1289) to RP. EV and JCB acknowledge their scholarship from CONACyT and IMSS. Authors acknowledge the Flow Cytometry Core Facility from ‘‘Coordinacion de Investigacion en Salud’’ of IMSS.
Supplementary Data Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j. arcmed.2016.12.004.
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