pRb and CyclinD1 Complement p16 as Immunohistochemical Surrogate Markers of HPV Infection in Head and Neck Cancer.

Identification of human papillomavirus (HPV) association in head and neck squamous cell carcinoma (HNSCC) is important to identify patients with favorable disease course. However, molecular HPV detection is not universally available. p16 has been proposed as a surrogate marker for HPV infection in HNSCC but, use on its own may result in wrong assignment of some cases to the group of HPV-associated tumors. We have therefore studied 424 HNSCC cases with known p16 and HPV DNA polymerase chain reaction (PCR) status for expression of retinoblastoma protein (pRb) and CyclinD1 by immunohistochemistry using 6-tiered scales (0 to 5) and a combined score (0 to 10). Sixty-one of 424 cases showed overexpression of p16. Of these, 52 cases were HPV DNA-PCR-positive. HPV association strongly correlated with low expression scores for pRb and CyclinD1 individually (scores ≤2) or combined (score sum ≤4), whereas HPV-negative carcinomas showed widely distributed expression scores. High expression scores for pRb or for pRb/CyclinD1 were observed exclusively in HPV DNA-PCR-negative cases. Three of 9 p16-positive/HPV DNA-PCR-negative cases showed high expression of pRb and displayed a high combined pRb/CyclinD1 score. We conclude that HPV-positive HNSCC are characterized by p16 overexpression and low scores for pRb, CyclinD1, and a low combined pRb/CyclinD1 score. High pRb or combined pRb/CyclinD1 scores are strong indicators for HPV-negativity and may justify excluding these cases from further molecular HPV testing. Furthermore p16-positive/HPV DNA-PCR-negative cases show heterogeneous expression of pRb and CyclinD1, including high pRb or high combined pRb/CyclinD1 scores suggesting that at least some of these cases are truly HPV negative.

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.

Relationship of Epstein-Barr virus and interleukin 10 promoter polymorphisms with the risk and clinical outcome of childhood Burkitt lymphoma.

Epstein-Barr virus (EBV) is an important environmental factor associated to the development of Burkitt lymphoma (BL) in endemic and intermediate risk regions. However, little is known about the contribution of genetic constitution to the development and clinical response of the disease. The aim of this work was to investigate the role of EBV and Interleukin 10 (IL10) single nucleotide polymorphisms (-1082A/G, -819C/T, -592C/A) and microsatellites (IL10.R and IL10.G) in susceptibility and clinical outcome in pediatric BL patients, in a region with intermediate EBV association frequency. The frequencies of IL10 promoter Single nucleotide polymorphisms -1082A/G, -819C/T, -592C/A, and IL10.R and IL10.G microsatellites were compared in 62 pediatric patients and 216 healthy donors. IL10 -1082GG and GCC/GCC genotypes were more frequent in patients than in controls, and associated to a higher risk of BL development (GG genotype OR 2.62, 95% CI, 1.25-5.51; P = 0.008; Pc = 0.024). EBV was detected in tumor samples by EBER-ISH in 54.1% of cases. EBV+ patients exhibited a better event free survival (EFS) (P = 0.019) than EBV- patients. Carriers of IL10 R3-GCC had worse EFS (P = 0.028). Our results suggest a risk effect and an independent prognostic value of IL10 polymorphisms and EBV in childhood BL patients.

Pediatric Hodgkin lymphoma in 2 South American series: a distinctive epidemiologic pattern and lack of association of Epstein-Barr virus with clinical outcome.

Hodgkin lymphoma (HL) shows a bimodal distribution with a first peak in developing countries during childhood. The causative role and prognostic significance of Epstein-Barr virus (EBV) association in patients with HL is controversial. Our aim was to perform a comparative study of EBV association in 2 Latin American pediatric HL series, and to correlate it with patient's survival. Epstein-Barr encoded RNAs in situ hybridization and latent membrane protein 1 immunohistochemistry were performed on formalin-fixed, paraffin-embedded HL biopsies from 176 pediatric patients from 2 public institutions from Argentina and Southeast Brazil. Mixed cellularity subtype was prevalent in Argentine HL (Arg HL) (52%) and nodular sclerosis subtype in Brazilian HL (BR HL) (83%). EBV expression was detected in 52% of cases, namely 54% Arg HL and 48% Br HL. EBV was significantly associated with mixed cellularity subtype in both populations. In Arg HL, EBV positivity was significantly higher in patients

Structural variability of the carboxy-terminus of Epstein-Barr virus encoded latent membrane protein 1 gene in Hodgkin's lymphomas.

Epstein-Barr virus (EBV) is implicated in the pathogenesis of several lymphoid and epithelial neoplasms. Latent membrane protein 1 (LMP1) is the major viral oncogene and it is controversial whether tumor LMP1 variants reflect their geographical predominance or are associated with enhanced oncogenic properties. This study aimed to analyze LMP1 molecular variability of 62 EBV+ Hodgkin's lymphomas and 22 non-neoplastic controls from Brazil and Argentina. EBV association was characterized by EBER-ISH, LMP1 immunohistochemistry and PCR assays for EBNA2 and 3C (typing), LMP1 30 bp deletion (del30) and number of 33 bp tandem repeats. LMP1 C-terminal sequencing was performed in 42 cases. EBV1 was the predominant strain in both geographical Hodgkin's lymphoma groups (average 82%). A higher frequency of del30 variant was observed in lymphomas (41/63) than in non-neoplastic controls (6/22) (OR 4.97, CI 95% 1.53-16.79; P = 0.005, chi(2) test). A large number (5-7) of 33 bp repeat units was characteristic of del30 LMP1 variants (P < 0.0001, Fisher's exact test). Sequence analysis showed a similar mutation spectrum to that described worldwide but none of the current classification schemes could be applied completely. A distinct structural pattern was observed in del30 variants, characterized by a large number of 33 bp repeat units and the presence of a 15 bp insertion encoding the JAK3 Box-1a motif (3/15 wt vs. 16/20 del30; P = 0.001, chi(2) test). The results suggest a pathogenic role for LMP1 del30 variants in Hodgkin's lymphoma from South America and point to particular virus-host molecular mechanisms, such as genomic instability in LMP1 carboxy-terminus, leading to enhanced production and selection of these deletion variants.

Timing of radiation in children with medulloblastoma/PNET.

BACKGROUND: To evaluate the importance of timing of radiation therapy in children with PNET/medulloblastoma, treated at the Brazilian National Cancer Institute (INCA). PROCEDURE: The records of 101 children with confirmed diagnosis of medulloblastoma were retrospectively reviewed. Patients had a median follow-up of 48 months (0.5-241 months). The age varied from 0.8 to 17.5 years (median: 7.6 years) and 21.7% were 3 years old or younger. RESULTS: According to the data collected from patients that received treatment for medulloblastoma from 1983 to 2001, the overall survival (OS) rate was 53% and the Disease Free Survival (DFS) rate was 40%. Multivariate analysis showed that under age 3 years, presence of neoplasic cells in the cerebrospinal fluid (CSF) at presentation or subtotal tumor resection resulted in a worse OS. The patients that received a biological effective dose (BED) greater than 44 Gy10 had better prognosis. Two-thirds of the patients had complete response after the initial treatment. Among them, 50% (34 patients) recurred, and of those 34 patients, 42% of them (14 patients) had recurrence in the posterior fossa. CONCLUSION: Surgery with total resection of the tumor and absence of neoplasic cells in the CSF are effective predictors of better OS. Radiotherapy was more effective when a BED was greater than 44 Gy10.