Pediatric cHL Research Articles

M1-like macrophage polarization prevails in young children with classic Hodgkin Lymphoma from Argentina

The microenvironment in classical Hodgkin lymphoma (cHL) comprises a mixture of different types of cells, which are responsible for lymphoma pathogenesis and progression. Even though microenvironment composition in adult cHL has been largely studied, only few groups studied pediatric cHL, in which both Epstein Barr virus (EBV) infection and age may display a role in their pathogenesis. Furthermore, our group described that EBV is significantly associated with cHL in Argentina in patients under the age of 10 years old. For that reason, our aim was to describe the microenvironment composition in 46 pediatric cHL patients. M1-like polarization status prevailed in the whole series independently of EBV association. On the other hand, in children older than 10 years, a tolerogenic environment illustrated by higher FOXP3 expression was proved, accompanied by a macrophage polarization status towards M2. In contrast, in children younger than 10 years, M1-like was prevalent, along with an increase in cytotoxic GrB+ cells. This study supports the notion that pediatric cHL exhibits a particular tumor microenvironment composition.

Plasma Thymus and Activation-Regulated Chemokine (TARC) As Diagnostic Marker in Pediatric Hodgkin Lymphoma

BackgroundPediatric classical Hodgkin lymphoma (cHL) is a clonal disorder in an inflammatory background, also known as the microenvironment. This microenvironment is of major importance for growth and survival of the malignant Hodgkin/Reed Sternberg (HRS) cells. HRS cells and the microenvironment communicate through chemo- and cytokines. Blood biomarkers result from this active crosstalk, and may be a surrogate for lymphoma viability (Steidl et al, JCC 2011). Blood biomarkers are important because they hold the promise to be easily available and cost-effective. One promising biomarker in adult patients with cHL is the “Thymus and Activation-Regulated Chemokine, TARC (Plattell et al, Haematologica 2012). Elevated TARC levels are also described in patients with atopic dermatitis (Hijnen et al, J All Clin Immunol 2004).In adult cHL patients about 85% of patients have significantly elevated levels of TARC in pre-treatment serum or plasma compared to healthy controls (Plattell et al).So far nothing is known about TARC in pediatric cHL patients. To define its value as a diagnostic marker in pediatric cHL patients, we compared TARC levels of pediatric cHL patients with control patients. This study was IRB-approved and registered under Dutch Trial registry number 6876.MethodsAfter providing informed consent, plasma and serum samples were collected of newly diagnosed cHL patients before start of treatment. To define normal values of TARC in children, samples were collected from non-cHL randomly selected patients from the hematology, endocrinology and oncology outpatient clinic. Baseline characteristics including history of atopic dermatitis were collected. These control patients were divided in three age groups (0-9,10-14 and 15-18 years). TARC levels were measured by enzyme-linked immunosorbent assay (R&D systems, Human CCL17/TARC Quantikine ELISA Kit). TARC levels of the cHL patients were compared to the control group to obtain ROC curves and calculate the AUC, cross-validated sensitivity and specificity and accuracy of TARC as a diagnostic marker. We hypothesized that pediatric cHL patients had elevated pretreatment TARC levels in both serum and plasma. Analyses were done using SAS V9.4.ResultsFourteen cHL patients were included with a median age of 14 (range 11-17) years. Ten (71.4%) were female. Eighty patients were included in the control group with a median age of 12 (range 10 months-18) years. Twenty-nine patients (36.3%) were included in age group 0-9, 25 (31.2%) in age group 10-14 and 26 (32.5%) in age group 15-18. Thirty-nine (48.8%) were female.Patients of the control group had a median TARC value of 71 (range 18-762) pg/ml for plasma and 318 (range 27-1300) pg/ml for serum. TARC plasma and serum levels decreased with age (Spearman correlation -0.26, 2-tailed p=0.0204), but there were no statistically significant pairwise comparisons found between the pre-specified age groups. In the eight control patients (10%) with atopic dermatitis no significantly higher plasma and serum levels were found (plasma median with eczema 97 versus 70 pg/ml without eczema (p=0.71) and serum median with eczema 643 versus 317 pg/ml (p=0.71)).Plasma was collected in 14 cHL patients, and all had elevated TARC levels, with a median plasma level of 18449 (range 1635-55821) pg/mL. Serum samples were collected in 8/14 cHL patients and all had elevated serum TARC levels. Median serum level: 46703 (range 12817-149739) pg/ml.The plasma TARC levels of cHL patients were significantly higher than those of the control group patients (p<0.001). With a cut-off of level of of 898.70 pg/ml, we obtained 100% (95% CI 73% - 100%) sensitivity and 100% (95% 94% - 100%) specificity. Serum TARC levels also were significantly higher than those of the control group patients (p<0.001), with a cut-off level of 10283.57 pg/ml, sensitivity and specificity will be 100% (95% CI 60% - 100% for sensitivity and 95% CI 94% - 100% for specificity).ConclusionAll classical cHL patients had significantly higher TARC levels compared to the 80 control patients. Despite the small sample size of cHL patients, TARC was found to be a sensitive and specific diagnostic marker for pediatric cHL in both plasma and serum. Further research with a bigger sample of cHL patients is necessary to improve the accuracy of the sensitivity, as well as to investigate whether TARC is also a valuable marker for disease response during treatment in pediatric patients with cHL. DisclosuresNo relevant conflicts of interest to declare.

Pathway‐focused gene expression profiles and immunohistochemistry detection identify contrasting association of caspase 3 (CASP3) expression with prognosis in pediatric classical Hodgkin lymphoma

The search for clinically relevant molecular markers in classical Hodgkin lymphoma (cHL) is hampered by the histopathological complexity of the disease, resulting from the admixture of a small number of neoplastic Hodgkin and Reed-Sternberg (H-RS) cells with an abundant and heterogeneous microenvironment. In this study, we evaluated gene expression profiles of 11 selected genes previously proposed as a molecular score for adult cHL, aiming to validate its application in the pediatric setting. Assays were performed by RT-qPCR from formalin-fixed paraffin-embedded (FFPE) lymph nodes in 80 patients with cHL. Selected genes were associated with cell cycle (CENPF, CDK1, CCNA2, CCNE2, and HMMR), apoptosis (BCL2, BCL2L1, and CASP3), and monocytes/macrophages (LYZ and STAT1). Despite using controlled preanalytical and analytical strategies, we were not able to validate the 11-gene score to be applied in pediatric cHL. Principal component analysis (PCA) disclosed 3 components that accounted for 65.7% of the total variability. The second PC included microenvironment and apoptosis genes, from which CASP3 expression was associated with a short time of progression-free survival, which impact was maintained in the unfavorable risk group, Epstein-Barr virus-negative cases, and multivariate analysis (P<.05). Because this is a counterintuitive association, CASP3 active expression was assessed at the protein level in H-RS cells by double immunohistochemistry. In contrast to the association of mRNA levels with a poor therapeutic response, a high number of cleaved CASP3+ cells were associated with longer progression-free survival (P=.03) and overall survival (P=.002). Our results demonstrate the feasibility of using FFPE samples as RNA source for molecular prognostication, but argue against the concept of direct and wide applicability of molecular scores in cHL. We reinforce the potential of CASP3 as an interesting target to be explored in adult and pediatric cHL, and alert for its dual biological role in H-RS cells and tumor microenvironment.

Macrophage Polarization Reflects T Cell Composition of Tumor Microenvironment in Pediatric Classical Hodgkin Lymphoma and Has Impact on Survival

Macrophages have been implicated in the pathogenesis of classical Hodgkin lymphoma (cHL) and have been suggested to have a negative impact on outcome. Most studies addressing the role of macrophages in cHL have relied on identification of macrophages by generic macrophage antigens, e.g., CD68. We have therefore conducted an in situ analysis of macrophage polarization in a series of 100 pediatric cHL (pcHL) cases using double staining immunohistochemistry, combining CD68 or CD163 with pSTAT1 (M1-like) or CMAF (M2-like). M1- or M2-polarised microenvironment was defined by an excess of one population over the other (>1.5). Expression of STAT1 and LYZ genes was also evaluated by RT-qPCR. Patients <14 years and EBV+ cases displayed higher numbers of CD68+pSTAT1+ cells than older children and EBV- cases, respectively (P=0.01 and P=0.02). A cytotoxic tumor microenvironment, defined by a CD8+/FOXP3+ ratio >1.5 was associated with higher numbers of CD68+pSTAT1+ (P=0.025) and CD163+pSTAT1+ macrophages (P<0.0005). Levels of STAT1 and LYZ expression were associated with the numbers of CD68+pSTAT1+ macrophages. EBV+ cHL cases disclosed a predominant M1 polarized microenvironment similar to Th1 mediated inflammatory disorders, while EBV- cHL showed a predominant M2 polarized microenvironment closer to Th2 mediated inflammatory diseases. Better overall-survival (OS) was observed in cases with higher numbers of CD163+pSTAT1+ macrophages (P=0.02) while larger numbers of CD163+CMAF+ macrophages were associated with worse progression-free survival (PFS) (P=0.02). Predominant M1-like polarization as disclosed by CD163+pSTAT1+/CD163+CMAF+ ratio > 1.5 was associated with better OS (P= 0.037). In conclusion, macrophage polarization in pcHL correlates with prevalent local T cell response and may be influenced by the EBV-status of neoplastic cells. Besides, M1-like and M2-like macrophages displayed differential effects on outcome in pcHL.

Pediatric Hodgkin Lymphoma in a South Indian Regional Cancer Center: Its Immunomorphology, Tumor-Associated Macrophages, and Association with Epstein–Barr Virus

Pediatric Hodgkin lymphoma (HL) comprises approximately a fifth of all patients with HL in India. Seventy-four cases of pediatric classical Hodgkin Lymphoma (cHL) from a regional cancer center in southern India were analyzed on a tissue microarray (TMA) for the stage of B-cell differentiation of the Hodgkin/Reed Sternberg (HRS) cell by immunohistochemistry (IHC) using CD10, bcl6, MUM1/IRF4, and CD 138. Fifty-two of seventy-four (70.3%) cases were of late germinal center/early post-germinal center phenotype (CD10−/bcl6−/MUM1+/CD138−). Epstein–Barr virus (EBV) association using Epstein-Barr virus encoded RNA (EBER) RISH and EBV-LMP1 immunohistochemistry (IHC) revealed an EBV association of 93%. Tumor-associated macrophages (TAM) in the microenvironment were also assessed on the TMA by CD68 IHC, and most cases (59.7%) showed >25% TAMs, with no case showing ≤5%. These findings indicate that pediatric cHL in India is a tumor, predominantly, of late germinal center/early post-germinal center B cells, is almost invariably EBV associated, and with a high number of TAMs in the microenvironment. This latter finding suggests that criteria other than TAM scores need to be developed for risk stratification of pediatric EBV-associated HL especially in developing countries.

PD1+ Cells in Pediatric Classical Hodgkin Lymphoma is Associated with Better Outcome

Classical Hodgkin lymphoma (cHL) is characterized by few neoplastic cells in a background of inflammatory cells. Many studies have described the T cell composition of tumour microenvironment in adult cases and we have demonstrated differences between pediatric and adult cHL in this respect. Two studies recently described that high numbers of PD1+ cells were associated with worse survival in adult cHL. PD1 is a receptor expressed by CD8+ and CD4+ T cells upon activation, as well as by T follicular helper cells, exhausted CD8+ T cells and effector memory CD8+ T cells. The objective of this study was to evaluate PD1+ cells in 100 paediatric cHL cases (3 to 18 y, median: 14 y) using immunohistochemistry and computer assisted microscopical analysis. The results were compared with the other T-cell populations as determined in our previous study of these cases. A median of 5 PD1+ cells/mm2 was observed (1 to 363 cells/mm2), while 40% of cases did not show any PD1+ cell. A direct correlation was observed between the numbers of PD1+ and CD4+ cells (P = 0.018), as well as PD1+ and CD8+ cells (P = 0.02). Higher numbers of PD1+ cells were observed in cases with cytotoxic microenvironment profile (P = 0.016), as disclosed by the ratio TIA1+ cells/FOXP3+ cells > 1.5. The numbers of PD1+ cells were not associated with age group or EBV-status. Cases with higher numbers of PD1+ cells (> 5 cells/mm2) were associated with better 5-years overall survival (P = 0.019). Our results suggest that the majority of PD1+ cells in the tumour microenvironment of paediatric cHL may contribute to the immune response against the neoplastic cells. A more detailed characterization of these cells is in progress.

Tumor-Associated Macrophages in Pediatric Classical Hodgkin Lymphoma: Association with Epstein-Barr Virus, Lymphocyte Subsets, and Prognostic Impact

Tumor-infiltrating macrophages are associated with adverse outcome in adult classical Hodgkin lymphoma (cHL). We have previously shown age-related changes in the lymphocyte composition of pediatric cHL. We therefore hypothesized that the number, function, and prognostic impact of macrophages in pediatric cHL would be different from adult cases. We analyzed the number of macrophages and dendritic cells (DC) in the tumor microenvironment of pediatric cHL by immunohistochemistry. Results were analyzed in context of age, histologic characteristics, Epstein-Barr virus (EBV) status, clinical follow-up, and our previous study of T-cell populations in these cases. One hundred cHL cases were studied, including 69% nodular sclerosis and 23% mixed cellularity cases. A total of 44.8% of cases were EBV-positive. Patients ≤10 years displayed more CD14(+) cells (P = 0.025). In comparison with nodular sclerosis, mixed cellularity was characterized by higher numbers of CD14(+), (P = 0.003) and CD163(+) cells (P = 0.027). EBV(+) cases exhibited higher numbers of CD14(+) (P < 0.0005), CD68(+) (P = 0.005), and CD163(+) cells (P = 0.02). CD68-positive cells did not display an effect on outcome. Worse overall survival was observed in cases with CD163/CD8 ratio ≥2 (P = 0.007). High numbers of CD163(+) cells were associated with worse progression-free survival (PFS; P = 0.015). Furthermore, high numbers of CD163(+) and granzyme B(+) cells were associated with worse PFS in EBV-negative (P = 0.005) but not in EBV-positive cases. Our results suggest that macrophage composition in pediatric cHL is distinct from adults. Functional status of macrophages and their value as prognostic indicators in pediatric cHL may depend on EBV status.

Highlights of This Issue

The cell-surface immunomodulatory protein B7-H3 is upregulated in a large proportion of human cancers. From a panel of B7-H3 monoclonal antibodies (mAb) generated via intact cell-based immunizations, Loo and colleagues selected a mAb displaying exquisite tumor/normal differential binding. MGA271, the resulting engineered anti–B7-H3 mAb, mediated potent antibody-dependent cellular cytotoxicity against a range of tumor types and showed potent antitumor activity toward tumor xenografts. The enhanced antitumor activity and favorable safety profile support initiation of a clinical study of MGA271 in patients with B7-H3-positive metastatic or recurrent adenocarcinoma.Tumor-infiltrating macrophages are potential independent indicators of poor outcome in classical Hodgkin lymphoma (cHL) in adults, but nothing is known in pediatric cases. In this study, Barros and colleagues investigated macrophages and dendritic cells in pediatric cHL, taking into account the Epstein–Barr virus (EBV) status of the Hodgkin and Reed–Sternberg cells and lymphocyte composition of the tumor microenvironment. The results show that macrophage composition in pediatric cHL is distinct from that of adult cHL and that the prognostic role of macrophages in pediatric cHL is affected by EBV status.Wong and colleagues performed a retrospective pharmacokinetic–pharmacodynamic analysis of xenograft/allograft efficacy data for 8 anticancer agents to investigate whether these preclinical models are predictive of clinical response. Tumor growth inhibition (TGI) was significantly correlated with clinical response only following simulation of TGI at clinically relevant exposures. Agents that led to greater than 60% TGI in preclinical models, at clinically relevant exposures, were more likely to lead to a clinical response. This study provides a framework for the successful use of murine subcutaneous tumor models to select active agents for further clinical evaluation.Madic and colleagues describe the validation in clinical samples of the pyrophosphorolysis-activated polymerization method to measure circulating tumor DNA (ctDNA) with a 1-copy sensitivity. ctDNA was shown in 20 of 21 patients with metastatic uveal melanoma in amounts correlated with the tumor burden. This technique, which is based on the detection of single-nucleotide mutations, is simple and inexpensive and could be implemented in any clinical molecular biology laboratory. The technique could also be applied to any tumor type with recurrent activating mutations, such as KRAS, BRAF, or PI3K.

Tumor microenvironment composition in pediatric classical Hodgkin lymphoma is modulated by age and Epstein‐Barr virus infection

Classical Hodgkin lymphoma (cHL) is characterized by a small number of neoplastic cells in a background of reactive cells. Children and adults differ in constitution and functionality of the immune system and it is possible that there may be age-related differences in tumor microenvironment composition in cHL. One hundred children with pediatric cHL were studied. Tumor-infiltrating lymphocytes were analyzed by immunohistochemistry (IHC) and image analysis. Epstein-Barr virus (EBV) status was determined by EBER-specific in situ hybridization and IHC. Results were analyzed in the context of age-group, histological characteristics and clinical follow-up. EBV-status was not associated with age-group. Children<10 years and EBV+ cases were characterized by a more intense T cell infiltrate, exhibiting a cytotoxic/Th1 profile, characterized by higher numbers of CD3+, CD8+, TIA1+ and TBET+ lymphocytes. Extranodal disease (p=0.016) and high number of GranzymeB+ lymphocytes (p=0.04) were independently associated with reduced progression-free survival (PFS). Yet, in EBV+ cases, improved outcome was observed in cases with low numbers of FOXP3+ lymphocytes (p=0.046), FOXP3/CD8 ratio<1 (p=0.021) and TBET/CMAF ratio<1 (p=0.017). By contrast, in EBV- cases, poor survival was observed in cases with extranodal disease (p=0.028), MC subtype (p=0.009) and high numbers of TIA1+ (p=0.044) and GranzymeB+ (p=0.04) lymphocytes. The results suggest that in EBV+ cHL an effective immune response directed against viral or tumor antigens may be triggered in the tumor microenvironment and that physiological and age-related changes of the immune system may also modulate the tumor microenvironment in pediatric cHL.

Disease patterns in pediatric classical Hodgkin lymphoma: a report from a developing area in Brazil

Epidemiological patterns established about 20 years ago, divided classical Hodgkin lymphoma (cHL) in three entities with regard to Epstein-Barr virus (EBV) status and histological subtypes and suggested different epidemiological patterns associated with degree of economic development. Here, we investigated histopathological features and EBV association in 100 consecutive pediatric cHL cases occurring in Rio de Janeiro (Brazil). Age at diagnosis ranged from 3 to 18 years (median 14 years) with 27% of cases ≤10 years. Unexpectedly, we did not observe an early childhood peak with most cases occurring in the >10 years age group. Nodular sclerosis (NS) was the most frequent subtype (69%) and was more frequently observed in the >10 years age group, followed by mixed cellularity (MC, 23%) which was distributed equally between age groups. EBV was identified in 44.8% of cases, without preferential association with age groups (≤10 years vs. >10 years). MC cases were independently associated with EBV infection of tumour cells (p = 0.045) and with a CD4/CD20 ratio <1 in the microenvironment (p = 0.014). Our results suggest that a gradual shift from childhood peak to early adulthood peak may be observed in developing regions. The development of MC subtype may result from early exposure to EBV in the context of an impaired immune system reflected by a CD4/CD20 ratio <1. Conversely, it is possible that NS originates predominantly in the context of a better immune response against EBV and/or tumour antigens expressed in the neoplastic cells.

The high frequency of EBV infection in pediatric Hodgkin lymphoma is related to the classical type in Bahia, Brazil

Pediatric Hodgkin lymphoma (HL) occurring in developing regions is different from HL in industrialized countries due to the higher frequency of association with Epstein-Barr virus (EBV) infection. This infection is related to classical HL (cHL) but is virtually absent in nodular lymphocyte predominant HL (nLPHL). We studied the phenotype and the expression of EBV gene products in 90 pediatric cases by immunohistochemistry and in situ hybridization. EBV-positive tumor cells were found exclusively in cHL. The infection occurred with high frequency in all cHL subtypes, but it predominated in the mixed cellularity and lymphocyte depletion subtypes. These results reinforce the hypothesis that EBV plays a major role in the etiology of pediatric cHL in developing areas. Curiously, the frequency of EBV infection in HL was identical to the previously described for Burkitt's lymphoma in the same pediatric population. As both lymphomas have a postulated precursor cell in the germinal center (GC), the pattern of latently EBV-infected GC cells previously described in Bahia may be related to the development of these lymphomas.