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.

Interleukin 10 (IL10) proximal promoter polymorphisms beyond clinical response in classical Hodgkin lymphoma: Exploring the basis for the genetic control of the tumor microenvironment.

Interleukin-10 (IL10) is an immune regulatory cytokine. Single nucleotide polymorphisms (SNPs) in IL10 promoter have been associated with prognosis in adult classical Hodgkin lymphoma (cHL). We analyzed IL10 SNPs -1082 and -592 in respect of therapy response, gene expression and tumor microenvironment (TME) composition in 98 pediatric patients with cHL. As confirmatory results, we found that -1082AA/AG; -592CC genotypes and ATA haplotype were associated with unfavourable prognosis: Progression-free survival (PFS) was shorter in -1082AA+AG (72.2%) than in GG patients (100%) (P = 0.024), and in -592AA (50%) and AC (74.2%) vs. CC patients (87.0%) (P = 0.009). In multivariate analysis, the -592CC genotype and the ATA haplotype retained prognostic impact (HR: 0.41, 95% CI 0.2-0.86; P = 0.018, and HR: 3.06 95% CI 1.03-9.12; P = 0.044, respectively). Our analysis further led to some new observations, namely: (1) Low IL10 mRNA expression was associated with -1082GG genotype (P = 0.014); (2) IL10 promoter polymorphisms influence TME composition;-1082GG/-592CC carriers showed low numbers of infiltrating cells expressing MAF transcription factor (20 vs. 78 and 49 vs. 108 cells/mm2, respectively; P< 0.05); while ATA haplotype (high expression) associated with high numbers of MAF+ cells (P = 0.005). Specifically, -1082GG patients exhibited low percentages of CD68+MAF+ (M2-like) intratumoral macrophages (15.04% vs. 47.26%, P = 0.017). Considering ours as an independent validation cohort, our results give support to the clinical importance of IL10 polymorphisms in the full spectrum of cHL, and advance the concept of genetic control of microenvironment composition as a basis for susceptibility and therapeutic response.

Prevalence of HPV infection in head and neck carcinomas shows geographical variability: a comparative study from Brazil and Germany.

Rising prevalence rates of high-risk human papillomaviruses (hrHPV) infection in oropharyngeal carcinoma (up to 80 %) have been reported in North America and Scandinavia. We have analysed 424 German and 163 Brazilian head and neck squamous cell carcinomas (HNSCC) from the oral cavity (OSCC), oropharynx (OPSCC) and hypopharynx (HPSCC) using p16 immunohistochemistry, HPV DNA PCR and sequencing, hrHPV DNA in situ hybridisation (ISH) and hrHPV E6/E7 RNA ISH. In the German series, 52/424 cases (12.3 %) were p16-positive/hrHPV-positive (OSCC 3.8 % [10/265], OPSCC 34.4 % [42/122], HPSCC 0 % [0/37]). In addition, there were 9 cases that were p16-positive/hrHPV-negative (5 OPSCC and 4 OSCC). In the Brazilian series, the overall hrHPV DNA prevalence by PCR was 11.0 % ([18/163]; OSCC 6 % [5/83], OPSCC 15.5 % [11/71], HPSCC 22.2 % [2/9]). Ten of these cases were hrHPV-positive/p16-positive. The remaining 8 hrHPV-positive/p16-negative cases were also negative in both ISH assays. Furthermore, 5 p16-positive/hrHPV-negative cases (2 OPSCC and 3 OSCC) were identified. In both series, HPV16 was by far the most common HPV type detected. We confirm that regardless of geographical origin, the highest hrHPV prevalence in HNSCC is observed in oropharyngeal carcinomas. The proportion of HPV-associated OPSCC was substantially higher in the German cohort than in the Brazilian series (34.4 vs. 15.5 %), and in both groups, the prevalence of hrHPV in OPSCC was much lower than in recent reports from North America and Scandinavia. We suggest, therefore, that it may be possible to define areas with high (e.g. USA, Canada, Scandinavia), intermediate (e.g. Germany) and low (e.g. Brazil) prevalences of HPV infection in OPSCC.

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.