Podoplanin: a Prognostic Marker and a Potential Target for Therapy in Bladder Cancer

Invasive bladder cancer is a frequent neoplastic diseases, and despite progresses made in early diagnosis and surgical procedures, the outcome of patients is characterized by high rate of mortality. This is mainly due to the lack of response to chemotherapy and radiotherapy, and other new resources are limited. In the present work we analyzed 50 consecutive cases of invasive bladder cancer treated by open surgery. Specimens were processed using standard histological procedures, including establishment of the histological diagnosis and grading. Additional slides were stained for podoplanin expression, clone D2-40 to investigate the structure and number of lymphatic vessels. We found lymphatic vessels in both intra- and peritumor areas, showing significant differences in morphology and number. Lymphatic invasion by tumor cells was higher in peritumor than in intratumor vessels. Lymphatic microvessel density correlated with stage and grade of the tumor. Of interest is the expression of podoplanin by tumor cells of urothelial carcinoma in about 14% of the cases, with strong correlation with grading. Based on our results, expression of podoplanin in invasive bladder cancer might indicate three potential targets: lymphatics, myofibroblasts, and tumor cells in selected cases. Keywords: bladder cancer, immunohistochemistry, podoplanin, therapeutic target

Cell-of-Origin (COO) Classification, BCL2 and MYC Expression Associated-Outcome in Younger Patients Treated By RCHOP Front-Line Therapy Versus Intensive Regimen Followed By Autologous Transplant for De Novo Advanced Diffuse Large B Cell Lymphoma (DLBCL) : Results of the French Prospective Multicenter Randomized Trial Goelams -075

Background: The prognostic value of COO classification by immunohistochemistry (IHC) for de novo untreated advanced DLBCL remains controversial after Rituximab-based frontline therapy. Other biomarkers such as BCL2 or MYC protein expression have been proposed to predict survival. IHC characteristics were investigated in a large multicenter randomized study. Methods: Three hundred twenty-three patients (pts) younger than 60 years with de novo untreated advanced DLBCL were randomized in the french prospective multicenter trial GOELAMS-075 to receive either 8 courses of RCHOP14 (n=161) or 2 courses of RCEEP (Rituximab, Cyclophosphamide, Eldisine, Epirubicine, Prednisone) and 1 course of Rituximab-Methotrexate-Cytarabine (RMC) followed by intensive BEAM conditionning with autologous transplant (ASCT) (n=162) upon negative interim PET-CT (visual analysis). In case of positivity, salvage regimen followed by ASCT was applied. Three years Event-free-survival (3y-EFS) was the primary endpoint. Event was defined by interim PET-CT positivity, progression or relapse, or death from any cause. Central pathology review confirmed de novo DLBCL diagnosis for 300 pts (93%). COO determination using Hans algorithm, BCL2 protein expression (clone 124, Dako) and MYC protein expression (clone Y69, Abcam) were recorded. Cut-off values were 70% for BCL2, and 40% for MYC. Results: COO analysis could be performed for 125/161 pts in RCHOP arm and 134/162 pts in intensive regimen arm including 36 and 34 Primary-Mediastinal-B-Cell subtype (PMBL) respectively. Repartition of non-PMBL was: 33/89 (37%) Germinal-Center subtype (GC), 56/89 (63%) Non-Germinal-Center subtype (NGC) in R-CHOP arm; 48/100 (48%) GC, 52/100 (52%) NGC in intensive regimen arm. Of 70 PMBL there were 50 NGC, 4 GC and 16 NE equally distributed in both arms. Clinical characteristics were similar in both GC and NGC subtypes, whereas PMBL presented with more frequent bulky disease and predominantly female gender. BCL2 ≥70% and MYC ≥40% were found in 147/285 (55%) and 85/185 (46%) of available samples, without difference between two arms. No correlation was found between BCL2 or MYC protein expression and GC or NGC subtype, however there were seen in a significantly lower proportion of PMBL (34% and 17% respectively). Coexpression of BCL2≥70% and MYC≥40% (MYC+/BCL2+) occurred in 52/184 (28%) cases, without difference between two arms or COO subtypes. By contrast, PMBL subtype displayed an extremely low rate of MYC+/BCL2+ cases (1/49, 2%). 3y-EFS rates were 52% ± 6% for GC, 58% ± 5% for NGC and 49% ± 6% for PMBL (p= 0,42) with no significant difference according to treatment arm. Of note, in PMBL, the majority of events was positive interim PET-CT. Worse EFS was seen in BCL2≥70% cases (3y-EFS: 47% ± 4% vs 60% ± 4%, p= 0,05) but this difference was erased in RCHOP arm (3y-EFS: 52% ± 6% vs 58% ± 6%). 3y-Progression Free Survival (PFS) rates were 73% ± 6% for GC, 76% ± 6% for NGC and 94% ± 4% for PMBL (p=0,03) with no difference between the two arms (Fig 1). There was no PFS difference in BCL2≥70% vs <70% cases (3y-PFS: 71% ± 4% vs 82% ± 4%, p= 0,11). EFS and PFS rates were similar between MYC≥40% and <40% cases (3y-EFS: 56% vs 59%; 3y-PFS: 78% vs 84%) without further advantage of one arm compared to another. Same results were obtained for MYC+/BCL2+ vs non MYC+/BCL2+ cases (3y-EFS: 53% vs 58%; 3y-PFS: 78% vs 81%). After a median follow-up of 71 months, PMBL was associated with significant better overall survival (OS) whereas no difference was observed between GC and NGC subtypes (5y-OS : 96% vs 75% and 78% respectively, p= 0,002) (Fig 2). OS rates were similar for BCL2 positive and BCL2 negative cases after exclusion of PMBL (5y-OS: 75% vs 78%, p=0,65). There was no significant impact of IHC MYC positivity (5y-OS : 80% vs 86% for MYC negative cases, p=0,29) or MYC+/BCL2+ coexpression (5y-OS : 80% vs 85% for negative cases, p=0,50) on outcome. There was no significant impact of treatment on OS of MYC and/or BCL2 positive cases. Conclusion: In younger patients, outcome of IHC defined GC and NGC subtype of non-PMBL DLBCL was not different following R-CHOP14 or intensive treatment including ASCT. Similarly, regardless of treatment arm, BCL2 or MYC or both overexpression did not impair significantly the prognosis. IHC defined COO or BCL2/MYC overexpression could not identify DLBCL in need of intensive therapy with ASCT. Finally the good prognosis of PMBL subtype with excellent PFS and OS was confirmed. Disclosures Cartron: Sanofi: Honoraria; Celgene: Honoraria; Gilead: Honoraria; Roche: Consultancy, Honoraria; GSK: Honoraria.

Virus-Specific Read-Through Codon Preference Affects Infectivity of Chimeric Cucumber Green Mottle Mosaic Viruses Displaying a Dengue Virus Epitope

ACucumber green mottle mosaic virus(CGMMV) was used to present a truncated dengue virus type 2 envelope (E) protein binding region from amino acids 379 to 423 (EB4). The EB4 gene was inserted at the terminal end of the CGMMV coat protein (CP) open reading frame (ORF). Read-through sequences of TMV or CGMMV, CAA-UAG-CAA-UUA, or AAA-UAG-CAA-UUA were, respectively, inserted in between the CP and the EB4 genes. The chimeric clones, pRT, pRG, and pCG+FSRTRE, were transcribed into full-length capped recombinant CGMMV transcripts. Only constructs with the wild-type CGMMV read-through sequence yielded infectious viruses following infection of host plant, muskmelon (Cucumis melo) leaves. The ratio of modified to unmodified CP for the read-through expression clone developed was also found to be approximately 1:1, higher than what has been previously reported. It was also observed that infectivity was not affected by differences in pI between the chimera and its wild counterpart. Analysis of recombinant viruses after 21-days-postinculation (dpi) revealed that deletions occurred resulting in partial reversions of the viral population to near wild type and suggesting that this would be the limiting harvest period for obtaining true to type recombinants with this construct.

Role of glial fibrillary acidic protein expression in the biology of human glioblastoma U-373MG cells

Object. The relationship between glial fibrillary acidic protein (GFAP) expression and glial tumor cell behavior has not been well defined. The goal of this study was to examine this relationship further. Methods. To investigate the relationship between GFAP expression and glial tumor cell behavior, the authors isolated clones from the human glioblastoma cell line, U-373MG, according to their level of GFAP expression. Immunochemical analysis demonstrated that one clone had consistently low GFAP expression (approximately 93% of cells were GFAP negative), whereas a second clone had consistently high GFAP expression (approximately 80% of the cells were GFAP positive). The structure, population doubling time, saturation density, anchorage-independent growth, migratory rate, and invasive potential of these two clones were determined in relation to their level of GFAP expression. Morphologically, both clones were composed of ameboid as well as stellate components. Although the population doubling times of the two clones were equally rapid, the clone with low GFAP expression demonstrated a slightly higher saturation density compared with the clone with high GFAP expression. In an anchorage-independent environment (soft agar), a greater difference in growth characteristics was noted between the two clones: the high-expression clone formed more colonies and these colonies were compact, well defined, and spherical, whereas the low-expression clone formed predominantly smaller, two-dimensional colonies with vague boundaries and isolated cells or groups of cells at the periphery. In contrast to these minor differences between the clones, the low-expression clone showed a markedly increased migratory rate and invasive potential compared with the high-expression clone. Therefore, the clone with reduced GFAP expression appeared more aggressive, demonstrating decreased contact inhibition, increased migratory rate, and increased invasive potential. Conclusions. These results suggest a direct correlation between GFAP expression and some measures of aggressive tumor growth and transformation properties.